DE10154540A1 - Procedure for predicting or predicting the effectiveness of tumor treatment - Google Patents

Abstract

A method for prediction or prognosis of the efficacy of a tumour treatment is disclosed, comprising an interaction partner and a therapeutic agent, the efficacy of which is predicted or prognosed by measuring a modulation of an EGF receptor-mediated signal. The invention further relates to a method of identification and modification of novel interaction partners and therapeutic agents for a tumour treatment.

Description

The present invention relates to a method for predicting or forecasting the Effectiveness of a tumor treatment that has an interaction partner and a Therapeutic includes and its effectiveness by measuring a modulation of an EGF receptor-mediated signal is predicted or predicted. In addition, the invention includes a method for identification and Modification of new interaction partners and therapeutics for tumor treatment.

Various documents are cited in the text of this description. The Disclosure content of the cited document (including all Manufacturer descriptions, information etc.) is hereby part of this description by reference.

While in the past infectious diseases have been the focus of medicine cancer today is the area in which the majority of medical research.

Every year 1.2 million new tumors of an invasive type are diagnosed. 560,000 Americans died of some form of cancer in 2000 (see "Epidemilogy: Reports find cancer rating declining "in" Health & Medicine Week ", 5-29, 2000).

The number of annual new diagnoses in women is from breast cancer and Men led by prostate cancer. When it comes to the number of deaths, the Lung cancer is the two entities with the largest number of new cases places two and three.

• a) lange Versuchszeiten (7-14 Tage);
• b) limitierte Auswertbarkeit durch die Gefahr von Kontaminationen;
• c) Bedarf an großen Tumorzellmengen;
• d) Notwendigkeit der Kultivierung von Tumorzellen.

The therapy of tumor patients represents a great challenge for the treating oncologist. The treatment spectrum of the doctor includes the surgical removal of the tumor, the radiation to the patient, and the administration of tumor-damaging agents. These agents are referred to below as therapeutic agents. Due to the individuality of tumors, no standard therapy that responds to all patients can be carried out to this day. In addition, the oncologist cannot rely on reliable tests that support him in the selection of one or more therapeutic agents for the treatment of a patient. The reason for the lack of such tests lies in the above-mentioned individuality of the tumors as well as the lack of usable functional test methods. All functional tests available today have the following disadvantages:

• a) long trial periods (7-14 days);
• b) limited evaluability due to the risk of contamination;
• c) need for large amounts of tumor cells;
• d) Need for the cultivation of tumor cells.

These restrictions can change the measurement results or the evaluation even make it impossible.

Various therapeutic agents are available for tumor treatment. This can be in cytostatics, cytokines, cytostatic hormones or Divide hormone derivatives and cytostatic antibodies. In Germany are 55 cytostatics, 8 cytokines, 17 hormones or hormone derivatives and 3 monoclonal antibodies approved as cancer therapeutics (Preiss et al. (2000), Oncology 2000: Recommendations for Therapy, 10th edition, W. Zuckschwerdt Verlag GmbH).

Breast cancer is the most common form of cancer in women and the second most common cancer-related cause of death after lung cancer. Heterogeneity in breast cancer brings with it the problem that standard therapies are hardly available. Tumors of one entity, but of different morphology, can vary greatly behave differently. To apply the most specific therapy possible prognostic and therapy-relevant factors have to be considered become.

Common procedures for tumor treatment today are surgery, radiation, Hormone and chemotherapy. These are often combined to increase the efficiency of the Improve treatment.

Chemotherapy is treated with cytostatics. There is one today Plenty of such substances, and for putting together the right ones Chemotherapy becomes the classic morphology and already the first "surrogate Marker ". Approx. 15 are used for breast cancer different treatments with cytostatics in question (Onkologie (2000) 10). Most of time These cytostatics have typical side effects because they are relatively unspecific Act. The increased metabolism of is used for the effect of the cytostatics Tumor cells. That is why chemotherapy means that rapidly growing cells like Immune cells or hair cells are also affected. The attack by immune cells manifests itself in an increasing immune deficiency under therapy, which often is the cause of therapy-associated complications.

Individual differences regarding the effect of the cytostatics have so far been possible usually do not clarify in advance. Therefore, in many cases, they become suboptimal Therapy forms selected.

A specific therapy that would target toxicity to tumor cells would be the approach that could reduce the occurrence of side effects. Therefore great efforts are being made to target tumor-specific "targets" identify which are characteristic of tumors, so-called tumor markers. This could be used on the one hand for diagnosis and on the other hand for therapy become.

Specific forms of therapy have already been described in the prior art require, for example, monoclonal antibodies that are specific to tumor cells, or target structures on their surface interact (Kischkel et al (1995), EMBO J., 14, 5579-88). As a result of such a specific interaction can Tumor cells can be eliminated by, among other things, antibody-induced signals. These antibody-induced signals are often signals that are generated by binding the specific antibodies to a receptor can be induced on the cell surface. The binding of an antibody can also, for example, the binding of an compete physiological ligands.

In this context, there is already one for receptors for growth factors Inhibition of the growth impulse described (Zimmermann et al. (2001) Microvasc. Res. 62, 15-25).

An example of a group of receptors for growth factors, their induced signal has been described as relevant in tumor development is the Family of receptors for epidermal growth factors (epidermal growth receptor family / EGFR family). For this receptor family have already been transmembrane members described. These proteins are intrinsic Tyrosine kinase activity. Examples of this are the EGFR (Her1, erbB-1; Ullrich et al. (1984), Nature 309: 418-425), Her2 (Her2 / new, erbB-2; Coussens et al., (1985), Science 230: 1132-1139), Her3 (erbB-3; Kraus et al. (1989), PNAS 86: 9193-9197; Guy et al. (1994), PNAS 91: 8132-8136) and Her4 (erbB-4; Plowman et al. (1993), PNAS 90: 1746-1750). These receptors are found in normal epithelia, mesenchymal, such as neuronal tissues expressed. Due to the fact that Receptors of this family were found to occur in some tumors postulates that this plays a role in the development and progression of the tumor Play tumor development (Cooke et al., (2001), European Journal of Cancer, 37: S3-S10).

The Her2 receptor appears to be of particular interest. This The receptor was first used for the rat (Shih et al. (1981), Nature 290: 261-264), later also for humans (Coussens et al. (1985) Science 230: 1132-1139) described. For human breast cancer, an overexpression of Her2 (King et al. (1985) Science 229: 974-976). This overexpression was also with a poor forecast, a shorter one Overall survival and an aggressive phenotype (Slamon et al. (1987) Science 235: 177-182). An overexpression of Her2 is in addition to breast cancers for colon cancers (Meltzer et al. (1987), Gastroenterology 92: 1174-1180), the lungs (Cline et al. (1987), Cancer 60: 2669-2674), the stomach (Jähne et al. (1994), surgeon 65: 307-311), the ovary (Meden et al. (1997), European Journal of Obstetrics & Gynecology and Reproductive Biology 71: 173-179), and des (Buchler et al. (2001), Journal of Gastrointestinal Surgery 5: 139-146). For this reason, Her2 is considered prognostic factor.

The transforming potential of Her2 was examined on NIH3T3 cells, which have been transfected with the Her2 gene. A moderate expression could do not induce any transforming activity during increased expression led to activation of the oncogene (Di Fiore et al. (1987), Science 237: 178-182). The EGFR group is a type I receptor tyrosine kinase (RTK). The According to the prior art, signal transmission takes place through a ligand-induced stabilization of a receptor dimer. The ligands of the EGFR Family have a bivalent character. A high affinity binding place with high Specificity for one receptor (EGFR, Her3, Her4) is at the N-terminal End and facilitates the binding of the low affinity binding site, its specificity is broader (Tzahar et al. (1997), The EMBO Journal 16: 4938-4950) and the Determines heterodimerization partner of the receptor (Tzahar and Yarden (1998) Biochim Biphys Acta, 1377, M25-27). Her2, which is only a low-affinity binding site for the physiological ligands described, interacts with receptors, which have a high affinity binding site, e.g. B. Her2 or Her4 (Horan et al. (1995) Journal of Biological Chemistry 270: 24604-24608).

Heterodimerization of the receptor induces intrinsic protein tyrosine Kinase activity and tyrosine autophosphorylation. These cellular events induce addition and phosphorylation according to the prior art various intracellular substrates (Steen H, et al. (2001) J Biol Chem. In press).

There are currently at least six ligands known for these receptors that are associated with bind different affinities to the receptors. Heregulin (HRG) is the most best investigated (indirect) ligand for the HER2 receptor. These ligands induce a specific one by binding to the receptors of the EGFR family Signal (EGFR-mediated signal).

Due to the high proliferation and metastasis rate of Her2 overexpressing cells (Her2 ⁺⁺ ) (aggressive phenotype (Cooke et al., (2001), European Journal of Cancer, 37: S3-S10)) Her2 was selected as a target for immunotherapy ,

The growth of human Her2 ⁺⁺ breast cancer cells has been shown to be inhibited with murine antibodies. Corresponding antibodies also inhibit growth in the natural environment of human tumors (Basalega et al. (1996), Journal of Clinical Oncology 14: 737-744; Lewis et al. (1993), Cancer Immunology, Immunotherapy 37: 255-263; Shepard et al. (1991) Journal of Clinical Immunology 11: 117-127).

An antibody with the described inhibitory function is from GENENTECH is marketed as Herceptin (trastuzumab). Although data about monotherapy with herceptin has been published (Cobleigh et al. (1999), Journal of Clinical Oncology 17: 2639-2648), the mAb is used today in combination with Cytostatics such as cisplatin used (Baselga et al. (1998), Cancer Research 58: 2825-2831; Pegram et al. (1999), Oncogene 18: 2241-2251). Herceptin is considered relatively well tolerated drug.

In addition, there are antagonists of the intracellular EGFR-mediated Pathways in clinical development. These are usually small chemical ones Compounds (such as quinazoline ZD1839 (Iressa ™) against tyrosine kinase of the EGF receptor (HER1).

Further studies with the above-mentioned mAb herceptin showed that the Effectiveness of Herceptin therapy from Her2 overexpression / gene amplification depends. For this reason, today is a positive Her2 receptor test Prerequisite for treatment with Herceptin.

From the FDA (Food and Drug Administration of the USA) are in this Relation to two test methods permitted (immunohistochemical staining (IHC) and fluorescence in situ hybridization (FISH)). A gene amplification can also with the help of the "Polymerase Chain Reaction" (PCR) or the "Southern Blot "(SB) can be detected. The expression of the protein can also with the help of an "enzyme-linked immunosorbation assay" (ELISA). These tests are a. in the work of Waldenmaier (establishment of a Her2 / new Receptor tests based on sensor-based technologies (2001), Diploma thesis from the Faculty of Biology at the Albert-Ludwigs-University, Freiburg im Breisgau) have been described in more detail and compared. On Disclosure content of this diploma thesis is hereby complete, e.g. T. preferred Embodiment of the method according to the invention described in more detail below, Referred.

According to IHC, patients are divided into four groups: 1. Status 0 and 2. 1+ classified as negative. The status 3. 2+ is considered slightly positive and the FDA recommends in In such cases, an additional FISH test to clearly determine the status. Status 4. 3+ is the IHC positive result. With FISH diagnostics the division into positive and negative cases, since the amplification is precisely defined can be. Although in patients overexpression of Her2 before appropriate therapy is proven, only 10 to 20% of these speak Patients on this expensive therapy (Baselga (2001), Annals of Oncology 12 suppl. 1: S49-S55).

A test that provides a clearer prognosis for the effectiveness of a Herceptin Therapy, or a therapy related to it, would be, also for reasons of Health economics, desirable. It is also for development new forms of therapy worth striving to find substances in combination with herceptine or similar medicines for effective tumor therapy are. Substances should also be identified in combination with known therapeutic agents as a corresponding or improved effectiveness Have herceptine and are preferably produced more cheaply can.

The present invention is therefore based on the technical problem Easy to use procedure that provides an improved prognosis about the efficacy of a therapy for tumors, which with a pathological expression of EGF receptors or of proteins involved in the Signal transduction of the corresponding receptor signal are involved in Connect.

This technical problem is characterized by that in the claims Embodiments solved.

• a) Inkontaktbringen einer Probe, die lebende Zellen mit einer abnormen Aktivität eines Mitglieds der EGF-Rezeptorfamilie aufweist mit
  • a) einem Interaktionspartner, wobei der Interaktionspartner das durch den natürlichen Liganden vermittelte Signal des Rezeptors moduliert; und
  • b) einem oder mehreren Therapeutika
  unter Bedingungen, die eine Interaktion der lebenden Zellen mit dem Interaktionspartner und dem dem einen oder mehreren Therapeutika erlauben; und
• b) Messung des über das Mitglied der EGF-Rezeptorfamilie vermittelten Signals, wobei die Modulation dieses Signals die Wirksamkeit der Tumorbehandlung prädiziert oder prognostiziert.

Accordingly, the present invention relates to a method for predicting or predicting the effectiveness of a tumor treatment

• a) contacting a sample that has living cells with abnormal activity of a member of the EGF receptor family
  • a) an interaction partner, the interaction partner modulating the signal of the receptor mediated by the natural ligand; and
  • b) one or more therapeutic agents
  under conditions that allow the living cells to interact with the interaction partner and the one or more therapeutic agents; and
• b) Measurement of the signal mediated by the member of the EGF receptor family, the modulation of this signal predicting or predicting the effectiveness of the tumor treatment.

The term "cells with abnormal activity of a member of the EGF- Receptor Family "describes cells in which the EGFR-mediated signal is changed physiologically (pathologically). This change includes both a ligand-induced change in the signal, as well as change that a Signal "further below" initiated in the signal path ("downstream"). This change can both an increase and an inhibition of the physiological signal his.

The "samples of living cells" are either for the invention Method according to that described in the description below Procedures isolated or derived from cultures of isolated patient cells (tissues). The term "interaction partner" is used in the context of the present invention Description of a molecule used that is specific to another molecule interacts. This interaction is by modulating the one of the characterized natural ligand-induced signal. The definition of Interaction partner includes organic as well as inorganic substances and Links. These are e.g. B. macromolecules and small molecules or low molecular weight molecules. The term also includes both chemical unmodified as well as modified molecules.

The term "macromolecules" refers to molecules with a high molecular weight Complexity or a high molecular weight understood. Are preferred this biomolecules such. B. biopolymers, especially proteins, oligomers or Polypeptides, but also DNA, RNA, oligo- or polynucleotides, prosthetic Groups, lipids, oligosaccharides and polysaccharides, and their modifications, as well also understood synthetic molecules. Come in connection with proteins ligands in particular, but also proteins or peptides, the epitopes or represent antigenic determinants of proteins. Furthermore, the Proteins can also be fusion proteins. The term includes peptides or proteins both natural and synthetic peptides or proteins. examples for natural proteins include a. Antibodies, antibody fragments, receptors that bind to their specific ligands, peptide ligands that bind to their specific receptors or peptide domains with specific substrates, including proteins and coenzymes, and other peptides or enzymes etc. to interact. Also included are forms of recombinant the aforementioned proteins or peptides. Natural peptides include accordingly including fragments of the proteins described above, with specific Affinity ligands interact. Include synthetic proteins or peptides both expressed pseudogenes or fragments thereof, and Proteins or peptides with a random amino acid sequence.

Under the term "small molecules" or "low molecular weight molecules" Understand molecules that are of less molecular complexity than those above defined macromolecules. In the literature, the term "small molecules" ("small molecules ") or" low molecular weight molecules " not used consistently. WO 89/03041 and WO 89/03042 describe molecules with molecular weights up to 7000 g / mol described as small molecules. Usually however, molecular weights between 50 and 3000 g / mol become more common between 75 and 2000 g / mol and mostly in the range between 100 and 1000 g / mol specified. Examples are known to the person skilled in the art from the documents WO 86/02736, WO 97/31269, US-A-5928868, US-A-5242902, US-A-5468651, US-A-5547853, US-A-5616562, US-A-5641690, US-A-4956303 and US-A-5928643.

Examples of such small molecules can be oligomers or small ones organic molecules such as oligopeptides, oligonucleotides, Carbohydrates (glycosides), isoprenoids or lipid structures. In the literature mostly the molecular weight is the basis for the definition of such small molecules represents.

The definition of the interaction partner thus includes molecules that have a specific Have interaction with a target molecule. Appropriate target molecules are Part of the EGFR-mediated signal transduction chain. With this Description includes both substances with extracellular domains (e.g. the receptors) or interact with intracellular target molecules. Examples for those intracellular target molecules that are part of the EGFR-mediated Signal transduction chain include inhibitors of kinases (e.g. Tyrosine Kinase Inhibitors, Protein Kinase C Kinase Inhibitors / PKC Kinase inhibitors) but also others, e.g. B. competitive substrates for this Signal transduction are relevant.

A specific interaction or interaction can, for example, with a "Key-lock principle" can be characterized. The interaction partner and that Target molecule, have structures or motifs that fit each other specifically, such as z. B. an antigenic determinant (epitope) associated with the antigen binding site Antibody interacts. Accordingly, there is a specific conflict to a more universal, non-specific interaction. By knowing the Structure of one of the interaction partners allows conclusions to be drawn about possible ones preferred structures or on special structural elements of a suitable one interacting partner.

The term "natural ligands" defines naturally occurring ligands that interact specifically with receptors of the EGFR family. examples for this are known to the person skilled in the art from the literature (Pinkas-Kramarski et al. (1997), J Mammary Gland Biol Neoplasia. 2 (2) 97-107)

The term "therapeutic agent" describes in the context of the present invention Substances (active substances) that are themselves used as medicinal products or as part of a can be administered. The term also includes Treatment methods used in tumor therapy. examples for such treatment methods are, for example, radiation therapy or hyperthermal forms of therapy.

The conditions that an interaction of the living cells with the Allow interaction partners and the therapeutic agent to ensure that a Interaction cannot be prevented by other components. This Components include e.g. B. components of culture media. This Conditions in the process according to the invention also include that other substances that may or may not have been demonstrated or only to a minor extent are effective.

The term "modulation" defines both in the context of this invention an increase, as well, in an alternative embodiment, an inhibition of the EGFR-mediated signal. Inhibition preferably also includes complete Block this signal.

In one embodiment of the method according to the invention, the modulation thereby achieved that the interaction partner instead of the natural ligand binds the receptor and thus prevents binding of the natural ligand or partially prevented (competed).

The effectiveness of the tested tumor treatment can be seen from the measured Derive modulation of the signal.

In a preferred embodiment of the method according to the invention, the Modulation of the receptor-mediated signal in a living cell with a abnormal activity of a member of the EGFR family modulating the corresponding and brought about under the same process conditions Signal in a living cell with normal activity of the member of the EGFR Family compared. The effectiveness of the tumor treatment is determined by the Difference in modulation predicts or predicts.

According to the definition described above for cells with an abnormal Activity of a member of the EGFR family are cells with normal activity of the Members of the EGFR family characterized in that the EGFR mediated Signal is not changed non-physiologically.

The same process conditions refer to the conditions under which the sample of the respective cells with the interaction partner and the Therapeutic agent can be brought into contact. The cells with the abnormal and the normal activity of the member of the EGFR family in parallel Approaches incubated according to the procedure described.

The therapeutic agent used in the method according to the invention is preferred selected from a group consisting of a substance with specific toxic effect, an ionic emitter, an antibody and an hyperthermic therapeutic.

The therapeutic agent is further preferably a combination of two or more Therapeutics.

The interaction partner and the therapeutic agent are also preferred Therapeutics merged together. The term "merged" describes in Connection with the invention, the coupling of the interaction partners with the Therapeutic / the therapeutic agents to a connection. It is accordingly Therapeutic in this preferred embodiment of the invention Procedure a substance.

• a) Inkontaktbringen einer Probe, die lebende Zellen mit einer abnormen Aktivität eines Mitglieds der EGF-Rezeptorfamilie aufweist mit
  • a) einem Interaktionspartner, wobei der Interaktionspartner das durch den natürlichen Liganden vermittelte Signal des Rezeptors moduliert; und
  • b) einer oder mehreren Testsubstanzen unter Bedingungen, die eine Interaktion der lebenden Zelle mit dem Interaktionspartner und der Testsubstanz erlauben;
• b) Messung des über das Mitglied der EGF-Rezeptorfamilie vermittelten Signals; und
• c) Vergleich des Meßergebnisses aus Schritt (b) mit einem Meßergebnis, das ohne die Zugabe der Testsubstanz erhalten wurde, wobei die Modulation dieses Signals mit der Testsubstanz im Vergleich zum Wert ohne die Testsubstanz ein Indiz für die Wirksamkeit der Testsubstanz als Therapeutikum oder als Leitsubstanz für ein Therapeutikum darstellt.

The invention further relates to a method for identifying a therapeutic agent effective for tumor treatment or a lead substance for such a therapeutic agent

• a) contacting a sample that has living cells with abnormal activity of a member of the EGF receptor family
  • a) an interaction partner, the interaction partner modulating the signal of the receptor mediated by the natural ligand; and
  • b) one or more test substances under conditions which allow the living cell to interact with the interaction partner and the test substance;
• b) measurement of the signal mediated by the member of the EGF receptor family; and
• c) Comparison of the measurement result from step (b) with a measurement result that was obtained without the addition of the test substance, the modulation of this signal with the test substance compared to the value without the test substance being an indication of the effectiveness of the test substance as a therapeutic agent or as a lead substance for a therapeutic agent.

The term "lead substance" describes in connection with the invention Substances that were identified using the method according to the invention. Such a lead substance is, however, in contrast to an identified one Therapeutic, formulated so that pharmacological parameters are optimized become. Parameters that are preferably optimized are described in the further description of Invention defined.

In a preferred embodiment of the method according to the invention Interaction partner a monoclonal antibody or a fragment or derivative from that.

Monoclonal antibodies and methods for their production are known to the person skilled in the art known. These are based on a first by Köhler and Milstein (1975) described method. This is u. a. in the Harlow and Lane laboratory manual (Antibodies, A laboratory manual; Cold Spring Harbor Laboratory; (1988); Chapter 6) described in detail. The definition also includes bispecific Antibodies, synthetic antibodies and fragments or derivatives of these antibodies. These include fragments such as Fab, Fv or scFv and chemically modified Derivatives of these antibodies or antibody fragments.

In a preferred embodiment, the monoclonal antibody is an IgG.

The monoclonal antibody is also preferably a humanized antibody.

In a further preferred embodiment of the invention The method is the humanized antibody Herceptin (trastuzumab).

• a) Inkontaktbringen einer Probe, die lebende Zellen mit einer abnormen Aktivität eines Mitglieds der EGF-Rezeptorfamilie aufweist mit
  • a) einem oder mehreren Tumortherapeutika; und
  • b) einem auf Wirksamkeit bei der Tumorbehandlung zu testenden Interaktionspartner, wobei der Interaktionspartner das durch den natürlichen Liganden vermittelte Signal des Rezeptors moduliert
  unter Bedingungen, die eine Interaktion der lebenden Zelle mit dem Tumortherapeutikum und dem Interaktionspartner erlauben;
• b) Messung des über das Mitglied der EGF-Rezeptorfamilie vermittelten Signals; und
• c) Vergleich des Meßergebnisses aus Schritt (b) mit einem Meßergebnis, das ohne die Zugabe des Interaktionspartners erhalten wurde, wobei die Modulation dieses Signals mit dem Interaktionspartner im Vergleich zum Wert ohne den Interaktionspartner ein Indiz für die Wirksamkeit des Interaktionspartners als Kombinationstherapeutikum oder als Leitsubstanz für ein Kombinationstherapeutikum darstellt.

The invention further relates to a method for identifying a therapeutic agent effective for tumor treatment or a lead substance for such a therapeutic agent

• a) contacting a sample that has living cells with abnormal activity of a member of the EGF receptor family
  • a) one or more tumor therapeutics; and
  • b) an interaction partner to be tested for efficacy in tumor treatment, the interaction partner modulating the signal of the receptor mediated by the natural ligand
  under conditions that allow the living cell to interact with the tumor therapeutic and the interaction partner;
• b) measurement of the signal mediated by the member of the EGF receptor family; and
• c) Comparison of the measurement result from step (b) with a measurement result that was obtained without the addition of the interaction partner, the modulation of this signal with the interaction partner compared to the value without the interaction partner being an indication of the effectiveness of the interaction partner as a combination therapeutic agent or as a lead substance for a combination therapeutic.

In a preferred embodiment of the methods described above, the Modulation of the receptor-mediated signal in a living cell with a abnormal activity of a member of the EGFR family modulating the corresponding signal in a living cell with normal activity of the members compared to the EGFR family. Here, the modulation of these signals in the different cells an indication of the effectiveness of the therapeutic agent or Combination therapeutic agent or a lead substance for such.

Preferably, the abnormal activity of a member of the EGFR family is due to one Overexpression of the receptor. Such overexpression includes expression a significantly increased number of the corresponding receptors on the Cell surface of the cells used. Also included is an elevated one Expression of the corresponding receptor without this on the surface of the Cells is expressed. This includes intracellular overexpression of the corresponding receptor.

In a preferred embodiment of the method according to the invention the interaction partner has intrinsic potential for modulating the Receptor-mediated signal.

In addition, the interaction partner is preferably an agonist of the Receptor-mediated signal. The interaction partner increase in this case as a result of Interaction with the specific target molecule the EGFR-mediated signal.

The interaction partner is also preferably an antagonist of the Receptor-mediated signal is. In this case, the interaction partner inhibits as a result of Interaction with the specific target molecule the EGFR-mediated signal. A such inhibition explicitly includes a complete blockage of the signal.

According to the method according to the invention, the member of the EGF Receptor family prefers the HER2 receptor.

The sample used for the method according to the invention is preferred a cell or tissue sample. Methods for isolating corresponding samples are known to the expert. This includes different types of withdrawal, at from which living cells are taken from a subject or patient. This are, for example, fine needle biopsies (fine needle aspirates), punch biopsies, the Removal of solid pieces of tissue (e.g. by surgical procedures), Pleural effusions, ascites samples, irrigation fluids, blood samples, urine samples, Sperm samples or samples of spinal and cerebral fluids or whole Organs (e.g. adrenal glands). The different removal techniques are e.g. B. described in: Kremer et al. (Surgical gauges, Georg Thieme Verlag, Stuttgart / New York (1999), Volume 4, p. 186; Volume 7, 2, p. 370; Volume E, Pages 146 to 147 and Volume 5, p. 238), Pichlmayr and Löhlein (Surgical Therapy, Springer Verlag (1991), pp. 225, 130, 89, 93, 116, 546 and 547), Malte (Angiology in clinic and practice, Georg Thieme Verlag (1998), p. 258), Niethard and Pfeil (orthopedics, Hippokrates Verlag Stuttgart, p. 38) and Bonk (biopsy and Operational preparation compendium for doctors and students).

The cell or tissue sample is preferably a tumor sample.

In a further preferred embodiment of the invention The tumor sample is a sample of a solid tumor or procedure metastatic cells derived from solid tumors.

This solid tumor is further preferably selected from a group comprising Breast cancer, ovarian cancer, bronchial cancer, colon cancer, Melanoma, bladder cancer, gastric cancer, head / neck cancer, brain cancer, Cervical tumors, prostate carcinomas, testicular carcinomas, swanomas, Leiomyosarcomas, bone tumors, renal carcinomas, pancreatic tumors and esophageal tumors.

The tumor sample is also preferably a sample of a tumor hematopoietic Origin. Tumors of hematopoietic origin are generically derived from hematopoietic stem cells. All are in accordance with this definition Lymphoma types included herein.

The tumor is preferably selected from a haematopoietic origin Group comprising malignant lymphomas, leukaemic cell, non-Hodgkin's Lymphomas, Hodgkin's lymphomas and thyroid lymphomas.

The tumor sample is also preferably a sample that circulates in the blood Tumor cells.

In a preferred embodiment of the method according to the invention, the Modulation of the receptor-mediated signal measured as induction of cell death or proliferation or inhibition of proliferation.

The induction of cell death includes both the induction of apoptosis (programmed cell death), as well as from necrosis.

This modulation is preferably measured in a functional assay. On The functional assay for the method according to the invention is preferably an assay in using intact, living cells (whole cell assays / whole cell assays). Assays in which preparations from cells or tissues (e.g. lysates) are produced, but are also encompassed by the described.

The functional assay is also preferably selected from a group including proliferation assays, cytotoxicity assays and metabolic assays.

Appropriate assays are known to the person skilled in the art. Examples of proliferation assays are assays that measure MTT or [ ³ H] thymidine incorporation. Cytotoxicity assays include both assays in which necrosis and / or apoptosis (see, for example, attached example 1) can be measured. Examples include DNA fragmentation tests (e.g. JAM test), [ ⁵¹ Cr] release tests, Annexin V tests, enzyme tests (e.g. caspase activation test), metabolic and morphological tests. The measurement can be carried out, inter alia, by flow cytometry or histologically. Examples of some of these cytotoxicity assays are summarized in Dulat et al. (Eur J Immunol. (2001), 31 (7), 2217-26).

Assay systems based on microphysiometer or are also included "Fluorescent Imaging Plate Reader (FLIPR) devices are based (Parce, (1989), Science, 246, 243-7; McConnel, I (1989) Science, 257, 1906-1912).

The measuring cavity is preferably designed in such a way that it has a plurality of internal ones Has surfaces. These are also preferably characterized by that one of these surfaces is a floor and that this is for the continuous Flow of solutions or suspensions is adjusted. This is preferred adapted that an unrestricted contact of the substance to be investigated and the cells is guaranteed and that the amount of contact with the cells coming substances can be controlled.

The measuring cavity in which the living cells are provided for the measurement is further preferably designed so that the cells in the chamber during a Flow of solutions or substances or other cell suspensions in this can be withheld. Restraining the cells in the Measuring cavity can be done in different ways. Preferably the Cells through spontaneous or natural adhesion of the cells to the inner surface retained in the chamber or by a porous membrane. Also preferred is a restraint by another type of carrier that is in the chamber is provided. It is also preferred to place the cells in a substrate, such as. B. gelatin, Embed collagen or agarose, fibronectin, poly-L-lysine. Also preferred is that depressions are provided at the bottom of the measuring cavity. In such The cells preferably remain in depressions. B. due to gravitational force even at an ordinary flow rate.

In a preferred embodiment, the measuring cavity is designed as described in EP-B1 0 394 406, in particular on page 4, lines 5 to 48, in FIGS. 1 to 4, and in the associated legends on page 6, lines 8 to page 7, Line 39, on page 8, lines 14 to 24 and page 8, line 44 to page 9, line 9. The solution or suspension can flow through the measuring cavity in accordance with process step (b) described there by means known to those skilled in the art. This includes pump systems or micropumps (e.g. for chromatography) that are common in laboratory technology. Preferably, a device should also be provided which ensures that the solution or suspension is free of air bubbles when introduced into the measuring cavity. The device for allowing the solution or suspension to flow through is preferably designed as described in EP-B1 0 394 406, in particular in FIGS. 5 and 6, and in the associated legends on page 5, line 40 to page 6, line 7, and on page 8, lines 1 to 13.

The measurement of the modulation of the receptor-mediated signal can also preferably using chemical, physical measurement methods. So can the pH z. B. by measuring the fluorescence or the absorption of a pH-sensitive dye can be determined (e.g. fluorescein or phenol red). The change in the redox potential can also be carried out using appropriate dyes be measured.

The next preferred embodiment of the method according to the invention concerns the therapeutic or combination therapeutic or the lead substance for such a therapeutic agent, which is a low molecular substance, a peptide, Aptamer, antibody or fragments or derivatives thereof. Definitions for the Groups of substances listed here have been described above or are known to the expert.

Another preferred embodiment of the method according to the invention gives suggests that this should be a high throughput process.

A particularly preferred embodiment shows that the invention Procedure is computer-assisted.

• a) die Bindungsstelle des Therapeutikums oder der Leitsubstanz für ein solches Therapeutikum an den Rezeptor, an einen rezeptorassoziierten Corezeptor oder an ein mit dem Signal des Rezeptors in Zusammenhang stehendes und signalvermittelndes Molekül identifiziert;
• b) die Bindungsstelle des Therapeutikums oder der Leitsubstanz für ein solches Therapeutikum an den Rezeptor, an einen rezeptorassoziierten Corezeptor oder an ein mit dem Signal des Rezeptors in Zusammenhang stehendes und signalvermittelndes Molekül durch molekulares Modellieren modifiziert; und
• c) das Therapeutikum oder die Leitsubstanz für ein solches Therapeutikum dergestalt modifiziert, daß dessen Bindungsspezifität oder Bindungsaffinität oder Bindungsavidität für den Rezeptor, einen rezeptorassoziierten Corezeptor oder ein mit dem Signal des Rezeptors in Zusammenhang stehendes und signalvermittelndes Molekül durch molekulares Modellieren erhöht wird.

A further particularly preferred embodiment relates to the method according to the invention as such which, in order to improve the pharmacological properties of the therapeutic agent or combination therapeutic agent identified according to the inventive method or the lead substance for such a therapeutic agent,

• a) identifies the binding site of the therapeutic agent or the lead substance for such a therapeutic agent to the receptor, to a receptor-associated coreceptor or to a signal-related molecule that is related to the signal of the receptor;
• b) the binding site of the therapeutic agent or the lead substance for such a therapeutic agent to the receptor, to a receptor-associated coreceptor or to a signal-related molecule which is related to the signal of the receptor and modified by molecular modeling; and
• c) the therapeutic agent or the lead substance for such a therapeutic agent is modified in such a way that its binding specificity or binding affinity or binding avidity for the receptor, a receptor-associated coreceptor or a signal-mediating molecule related to the signal of the receptor is increased by molecular modeling.

Another particularly preferred embodiment relates to an inventive one Method in which the binding sites in step (a) are determined by position-specific Mutagenesis can be determined.

The identified or improved therapeutic agent or is further preferred the lead substance for such a therapeutic agent through peptidomimetics pharmacologically further improved.

The next particularly preferred embodiment focuses on Process according to the invention which is used to improve the pharmacological Properties of the inhibitor identified by the method according to the invention or the inhibitor improved by the method according to the invention, however, the polypeptide is further modified as a lead structure to a modified one active center, a modified spectrum of activity, a modified Organ specificity, improved activity, increased toxicity to Tumor cells (an improved therapeutic index), decreased Side effects, a delayed start of therapeutic effectiveness or length of therapeutic efficacy, changed pharmacokinetic Parameters (absorption, distribution, metabolism or excretion), modified physicochemical parameters (solubility, hygroscopic properties, color, Taste, smell, stability, condition), improved general specificity, Organ / tissue specificity, and / or an optimized administration form and -route shows what is caused by the esterification of carboxyl groups, hydroxyl groups with carboxylic acids, hydroxyl groups, for example phosphates, pyrophosphates, Sulfates, "hemisuccinates" or the formation of pharmaceutically acceptable Salts, pharmaceutically acceptable complexes or the synthesis of pharmacologically active polymers or the introduction of hydrophilic groups, the introduction or exchange of substituents in aromatics or Side chains, changing the substituent pattern or modification through the introduction of isosteric or bioisosteric groups or the Synthesis of homologous compounds, or the introduction of branched ones Side chains, the conversion of alkyl substituents to cyclic analogues, the Derivatization of hydroxyl groups to ketals or acetals, the N-acetylation to amides, phenyl carbamates, the synthesis of Mannich bases or imines or through the conversion of ketones, aldehydes into ship bases, oximes, acetals, Ketals, enol esters, oxaholidines, thiozolidines or combinations thereof becomes.

Another particularly preferred embodiment relates to the invention Process for the manufacture of a medicinal product or medicinal product a therapeutic agent obtained by the method according to the invention or the Lead substance for such a therapeutic agent with a pharmaceutically acceptable Carrier or diluent is formulated.

In addition, the therapeutic agent or combination therapeutic agent is preferred or the lead substance for such a therapeutic agent is a cytostatic agent or cytotoxic substance.

In another embodiment, the invention relates to the use of an after the therapeutic agent identified according to the method of the invention or Combination therapeutic agent for the manufacture of a medicament or Medical device for the treatment of tumors and or tumor diseases.

The invention further relates to a composition comprising one or more Contains therapeutics or combination therapies that after the Processes according to the invention have been identified or improved.

The figures show:

Fig. 1 shows a schematic representation of the HER2 / neu receptor: two extracellular cysteine-rich domains and two hydrophilic regions flanking the tyrosine kinase domain, determine the characteristics of the receptor. Peles et al., 1993;

Fig. 2, the interaction of the receptors of the EGF family are dependent upon the ligand and the receptor Lung assembling the cell. A: Heregulin (HRG / NDF / NRG) binds the HER3 (3) and HER4 (4) receptors. They then prefer to recruit HER2 / neu receptors (2). If there is no HER2 / new on the cell membrane, the HRG receptors interact with EGFR receptors (HER1) (1). B: EGF is shown as an example for the group of EGFR-binding molecules. This binds EGFR. He can then dimerize with the other members of the EGFR family, preferably with HER2 / neu. C: BTC is shown as an example for the group of EGFR and HER4 binding molecules. This binds EGFR and HER4. These receptors can then interact preferentially with HER2 / new but also with HER3. Graus-Porta et al., 1997;

Fig. 3 Effect of Herceptin monotherapy in metastatic, HER2 / neu positive breast cancer (n = 43, HER2 / neu +): The response of the tumor was examined after 11 weeks at the end of the first treatment period. If the tumor could not be detected with visual and X-ray examinations after this time, this was defined as complete remission. If a reduction in tumor size of over 50% was observed, this was described as a partial remission. With a low remission, the tumor was reduced by over 25%, while the term "stable disease" describes the condition without changing the tumor size. Baselga et al., 1996;

Fig. 4 Summary of two phase III studies on the combination of AC and paclitaxel with Herceptin. Baselga et al., 2001;

Fig. 5 Comparison of methods for HER2 / neu diagnostics. Müller et al., 2000;

Fig. 6 HER2 / new diagnostics: influence of the inclusion size on the result. Bojar, H. et al. 1998;

Fig. 7 cellular biology and extracellular acidification. Mc Connell et al., 1992;

Fig. 8 medium metabolic activity of different cell lines, depending on the cell density;

Fig. 9 Stimulation of SK-BR-3 cells with different concentrations of HRG stimulation in an interval of 30 minutes;

Fig. 10 dose response curve SK-BR-3: Mean and standard deviation of various HRG stimulation experiments with a stimulation interval of 30 minutes (n = 8);

Fig. 11 Stimulation of MDA-MB-453 cells with different concentrations of HRG stimulation in an interval of 30 minutes;

Fig. 12 dose response curve MDA-MB-453: Mean values and standard deviation of various HRG stimulation experiments with a pacing interval of 30 minutes (n = 8);

Fig. 13 Comparison of the dose-response curves of the cell lines SK-BR-3 and MDA-MB-453: Mean values and standard deviation of various HRG stimulation experiments with a pacing interval of 30 minutes (n = 8);

Fig. 14 stimulation of MCF-7 cells with different concentrations of HRG stimulation in an interval of 30 minutes;

Fig. 15 Stimulation of SK-OV-3 cells with different concentrations of HRG stimulation in an interval of 30 minutes;

Fig. 16 Stimulation of SK-BR-3 cells with 100 ng / ml HRG at different exposure time;

Fig. 17 Stimulation of MDA-MB-453 cells with 100 ng / ml HRG at different exposure time;

Figure 18 Partial inhibition of HRG stimulation by Herceptin in SK-BR-3;

Fig. 19 Relative HRG stimulation depending on the Herceptin concentration: dose-response curve SK-BR-3 (n = 4);

Figure 20 Inhibition of HRG stimulation by Herceptin in MDA-MB-453;

Fig. 21 Inhibition of HRG stimulation (30 minutes) in SK-BR-3 by the tyrosine kinase inhibitor genistein;

Fig. 22 Influence of solvent on paclitaxel chemosensitivity measurements SK-BR-3 cells;

Fig. 23 Influence of solvent on paclitaxel chemosensitivity measurements in MDA-MB-453 cells;

Fig. 24 averages and standard deviations for the chemosensitivity of SK-BR-3 relative to the cytostatic agent cisplatin and Herceptin (n = 5);

Fig. 25 averages and standard deviations for the chemosensitivity of SK-BR-3 relative to the cytostatic carboplatin and Herceptin (n = 5);

Fig. 26 averages and standard deviations for the chemosensitivity of SK-BR-3 relative to the cytostatic vinorelbine and Herceptin (n = 5);

Fig. 27 averages and standard deviations for the chemosensitivity of SK-BR-3 relative to the cytostatic Gemcitabine and Herceptin (n = 5);

Fig. 28 compiling the profile of action and effects of Herceptin on SK-BR-3 cells: mean values of the end points;

Fig. 29 averages and standard deviations for the chemosensitivity of MDA-MB-453 with respect to the cytostatic agent cisplatin and Herceptin (n = 5);

Fig. 30 averages and standard deviations for the chemosensitivity of MDA-MB-453 with respect to the cytostatic carboplatin and Herceptin (n = 5);

Fig. 31 averages and standard deviations for the chemosensitivity of MDA-MB-453 with respect to the cytostatic vinorelbine and Herceptin (n = 5);

Fig. 32 averages and standard deviations for the chemosensitivity of MDA-MB-453 with respect to the cytostatic Gemcitabine and Herceptin (n = 5);

Fig. 33 Summary of the effect profile and effects of Herceptin on MDA-MB-453 cells: mean values of the end points;

Fig. 34 averages and standard deviations for the chemosensitivity of MCF-7 with respect to the cytostatic agent cisplatin and Herceptin (n = 5);

FIG. 35 means and standard deviations for the chemosensitivity of MCF-7 with regard to the cytostatic carboplatin and Herceptin (n = 5);

Fig. 36 averages and standard deviations for the chemosensitivity of MCF-7 with respect to the cytostatic vinorelbine and Herceptin (n = 5);

Fig. 37 averages and standard deviations for the chemosensitivity of MCF-7 with respect to the cytostatic Gemcitabine and Herceptin (n = 5);

Fig. 38 Compilation of the effect profile and effect of Herceptin on MCF-7: mean values of the end points;

Fig. 39 averages and standard deviations for the chemosensitivity of SK-OV-3 with respect to the cytostatic agent cisplatin and Herceptin (n = 5);

Fig. 40 averages and standard deviations for the chemosensitivity of SK-OV-3 with respect to the cytostatic carboplatin and Herceptin (n = 5);

Fig. 41 averages and standard deviations for the chemosensitivity of SK-OV-3 with respect to the cytostatic vinorelbine and Herceptin (n = 5);

Fig. 42 averages and standard deviations for the chemosensitivity of SK-OV-3 with respect to the cytostatic Gemcitabine and Herceptin (n = 5);

Fig. 43 Compilation of the effect profile and effects of Herceptin on SK-OV-3 cells: mean values of the end points;

Fig. 44 Average increase in metabolic activity after 30 minutes of exposure to 100 ng / ml HRG in different cell lines.

The following examples illustrate the described invention.

example 1 Activity, proliferation and apoptosis

Different cell lines (KB, MDA-MB-453, MCF-7, SK-BR-3, SK-OV-3) were used in the ChemoSelect® test analyzed on various cytostatics. The determined Efficacies stood with the tests for proliferation and for apoptosis were measured in relation. This makes it possible to make predictions about the Make therapeutic efficacies based on laboratory results. In particular, the analysis of patients showed that cytostatics, with the largest Effects in the laboratory (ChemoSelect®), also the best healing success in the clinic showed.

Example 2 Ligand / target functionality

The cell lines MDA-MB-453, MCF-7, SK-BR-3, SK-OV-3 were designed on the Functionality of the ligand HRG tested. It was found that only the Cell lines that express HER2 and either HER3 and / or HER4 on HRG responded at all (SK-BR-3 and MDA-MB-453), while one only HER2 (SK- OV-3) and a low HER2-expressing cell line (MCF-7) showed no activity. These results were also confirmed on patient material. So there is a direct test for the functionality of target molecules.

Example 3 Therapeutic / target functionality

Only the HRG-sensitive cell lines used in Example 2 (SK-BR-3 and MDA-MB-453) showed a reduction in HRG stimulation with the anti-HER2 Antibodies (trastuzumab) while the negative controls (SK-OV-3 and MCF-7) - as expected - had no effect on trastuzumab treatment. This Results were also obtained from patient material and correlated with that Response to trastuzumab therapy.

Example 4 combination therapies

Treatment of the cell lines used in Example 2 gave trastuzumab or with a cytostatic (vinorelbine, cisplatin, gemcitabine, carboplatin) alone no great therapeutic success. When combining trastuzumab with one The cytostatic agent, however, showed clear synergy effects. This included the combination of vinorelbine or cisplatin with trastuzumab in the SK-BR-3 cell line and vinorelbine, gemcitabine or carboplatin with trastuzumab on the MDA-MB-453 Cell line. The two negative controls (SK-OV-3 and MCF-7) again showed how hardly expects synergy effects. The correlation between this type of functional Diagnosis and a number of patients showed good agreement.

Example 5 More interaction partners

The tests necessary for this point are the same Experimental arrangements from example 2. It shows that HRG only in Presence of another EGF receptor interacts with HER2. For this are the decisive tests the negative controls, which show that only one complete set of receptors (e.g. HER2 and HER3) the ligand the signal into the Cell can pass on. These data also correlate with the patient data.

Example 6 Establishment of a Her2 / neu receptor test based on sensor-based technologies 1 Material and methods 1.1 cell lines and cell culture

The cell lines MDA-MB-453 (Cat. No .: ACC 65) and MCF-7 (Cat. No .: ACC 115) were from the German Collection of Microorganisms and Cell Cultures (DMSZ, Braunschweig, Germany). The SK-BR-3 cell lines (Cat. No .: HTB-30) and SK-OV-3 (Cat. No .: HTB-77) were from the "American Type Culture Collection "(ATCC, Manassas, USA).

The cell lines SK-BR-3 and SK-OV-3 were in modified Mc Coy's 5A medium with L-glutamine (LIFE TECHNOLOGIES, cat.no .: 26600-023), 10% FCS (PAA, Cat. No .: A 15-043) and 100 units / ml penicillin G, 100 µg / ml streptomycin Cultivated sulfate (LIFE TECHNOLOGIES, Cat.No .: 15140-122).

The cell line MDA-MB-453 was in Leibowitz L-15 medium with L-glutamine (LIFE TECHNOLOGIES, Cat. No .: 11415-056), 10% FCS (PAA, Cat. No .: A 15-043) and 100 units / ml penicillin G, 100 µg / ml streptomycin sulfate cultivated (LIFE TECHNOLOGIES, Cat.No .: 15140-122).

The cell line MCF-7 was in RPMI 1640 medium with L-glutamine (PAN, Cat. No .: P 04-16500), 10% FCS (PAA, Cat. No .: A 15-043), 100 units / ml penicillin G, 100 µg / ml Streptomycin sulfate (LIFE TECHNOLOGIES, Cat. No .: 15140-122), MEM non-essential amino acids (LIFE TECHNOLOGIES, Cat. No .: 11140-035), 1 mM Sodium pyruvate (GIBCO, Cat. No .: 11360-039) and 10 µg / nl bovine insulin (BIOCHROM, cat.no .: K 3510) cultivated.

Cells were washed every 2-3 days with 2-3 ml of 1 × trypsin-EDTA solution (LIFE TECHNOLOGIES, Cat.No .: 45300-043) and in a ratio of 1: 2 passaged. Each time before use for experiments, the cells were 2-3 ml EDTA / EGTA solution replaced. This consisted of 0.25 mM EDTA (ROTH, Cat. No .: 8043.1), 0.125 mM EGTA (ROTH, Cat. No .: 3054.2), 30 mM Tris (ROTH, Cat. No .: 4855.2) 0.25 M sucrose (ROTH, Cat. No .: 4621.1) and was subsequently used NaOH (ROTH, Cat. No .: 6771.1) adjusted to a pH of 7.8.

1.2 Freezing culture cells

Reserve samples were formed from passages P3 to P8. The cells were detached with trypsin as described above. Approx. 10 ⁶ cells each were mixed with 2 parts of the respective medium, 2 parts FCS (PAA, Cat. No .: A 15-043) and one part of DMSO (SIGMA, Cat. No .: D2579) in 2 ml cryotubes (COSTAR ) given. The cells were cooled for 2-4 hours at 4 ° C and overnight at -70 ° C before being stored in liquid nitrogen.

1.3 Cytostatics

Drugs that show synergistic or additive effects in combination with Herceptin have been used according to their "peak plasma" concentration. Substances with different mechanisms of action were selected:
Antimetabolites: gemcitabine (22.5 µg / ml; LILLI, Germany)
Mitosis inhibitors: vinorelbine (1 µg / ml; PIERRE FABRE, Germany), paclitaxel (13.6 µg / ml, BRISTOL MYERS, Germany)
Alkylating agents: carboplatin (8 µg / ml; SIGMA, Germany), cisplatin (3.8 µg / ml; SIGMA, Germany)

6 mg paclitaxel was dissolved in 0.5 ml cremophor and 0.5 ml 100% EtOH. All other cytostatics used were water soluble.

The Paclitaxel solvent control consisted of 1.13 µl / ml Cremophor and 1.13 µl / ml 100% EtOH together.

1.4 Consumables

Human Serum Albumin (HSA) was developed by SIGMA-ALDRICH (Germany) related (Cat. No .: 05420). The recombinant EGF domain of human HRG-α was obtained from R & D SYSTEMS (Minneapolis, USA). A stick solution one Concentration of 50 µg / ml was, as recommended, with 0.1% HSA based on PBS manufactured. The anti-HER2 / neu monoclonal antibody trastuzumab (Herceptin) was obtained from ROCHE DIAGNOSTICS (Mannheim, Germany). The tyrosine kinase inhibitor genistein (Cat. No .: G 6776) was developed by SIGMA ALDRICH (Germany) related.

1.5 buffer solutions

Modified "low buffered" RPMI 1640 medium with L-glutamine and without NaHCO ₃ (Pan, Cat. No .: P 04- 162000) was used as the running buffer for cytometric preliminary measurements.

Further experiments were carried out with a "Balanced Salt Solution" (BSS). This consisted of 1 M glucose (ROTH, cat. No .: 6887.1), 4 M NaCl (ROTH, cat. No .: 3957.1), 1 M KCl (ROTH, cat. No .: 6781.1), 1 M CaCl ₂ (ROTH, Cat. No .: 5239.1), 1 M MgCl ₂ (ROTH, Cat. No .: 2189.1), 1.1 M NaH ₂ PO ₄ (ROTH, Cat. No .: 2370.1) and 0.5 M Na ₂ HPO ₄ (ROTH, Cat. No .: 4984.1) together and was adjusted to a pH of 7.3 with Na ₂ HPO ₄ .

1.6 Measurement of Tumor Cell Activity Using the Cytosensor Microphysiometer

The microphysiometer used is from MOLECULAR DEVICES INC. marketed as a cytosensor. It represents a biological application from the Computer technology known silicon technology. With the help of this Effects of various substances on the biological can be applied Represent the function of cells. A common application for the cytosensor is Investigation of receptor-ligand interactions.

The energy requirement of cells in culture is primarily met by glycolysis. This conversion of glucose and oxygen into intermediate products such as protons, lactate and CO ₂ leads to an acidification of the surrounding medium. This acidification rate is the central measure of the cytosensor microphysiometer and characterizes the cell metabolism ( Fig. 7). The metabolism is influenced by substances such as toxins, hormones, viruses or cosmetics and can be measured with the help of the cytosensor.

The central unit of the cytosensor microphysiometer (cm) is a LAPS ("Light- Addressable Potentiometric Sensor "). This unit measures the Concentration of certain ions that represent the surface potential at the interface between an electrolytic solution and the surface of the semiconductor. The The surface of the LAPS consists of a 130 nm thin oxynitrite insulating layer. This separates the silicon chip from the aqueous medium. By a Reference electrode can apply a voltage to the interface of the sensor create an electric field oriented perpendicular to it. One on the Underside of the sensor-directed LED light source emits photon pulses, which are absorbed by the silicon layer, which leads to the formation of Leads electron hole pairs. Since they move in the electric field, they are measurable as a temporary current. The resulting alternating current is dependent of the applied pre-tension and of the surface tension of the Silicon chips. The oxynitrite layer can bind protons in solution, which creates an additional electrostatic potential. The AC is therefore pH dependent. Such a measurement will result in a Sensitivity of over 1/1000 pH units reached.

In the measuring unit of a LAPS, cells are placed on a permeable one Polycarbonate membrane introduced. Adherent cells are opened directly this membrane is cultivated during cell suspensions using agarose be immobilized. This measuring unit flows, driven by a Peristaltic pump, a fluid flow that the cells on the one hand Nutrients supplied, on the other hand metabolites removed. To be flowed around the cells in this case from a suitable medium that supplies them with essential substances guaranteed, but in contrast to culture medium one has significantly lower buffer capacity. Here it has become the standard medium a modified RPMI medium (PAN) proves that which Sodium bicarbonate system is missing. Experiments have shown that a "balanced salt Solution "(BSS, see section 1.5) can increase the resolution of the sensor. Cells can be observed in cm for hours or days. The Perfusat can Substances are added to their effect on the cells used characterize. The periodically Liquid flow interrupted and the acidification of the medium as a measure of the cellular metabolic activity measured. That way, depending on specific effects can be observed by using the Metabolism as a result of individual measurements is recorded. Application examples are toxicity studies of cytostatics Tumor cells, compatibility tests with cosmetics or specific receptor Ligand reactions.

In addition to the peristaltic pumps and the LAPS, there are degassers ("debubblers"), Heating systems and controllable valves are important components of the cm. The valves allow switching of liquid flow from a reference or Calibration medium to the medium provided with the substance to be examined from a separate container. The time of switching can, as well how the duration of exposure can be arbitrarily determined. An integrated Heating system determines the temperature of the medium between 4 ° C and 43 ° C. A unit of one cm consists of eight individual channels and a central one Computing unit that controls the system.

1.6.1 Stimulation of the Tumor Cells with HRG

Before plating, the cells were detached from the culture flasks with EDTA / EGTA dissociation solution (see section 1.1) and in the case of the SK-OV-3 cell line using a cell scraper. The cells were then left on the polycarbonate membrane for growth for at least 10 h at 37 ° C. and 5% CO ₂ in an appropriate medium with 10% FCS. Immediately before the measurement in cm, this culture medium was exchanged for a corresponding medium without FCS. The cells were left in this medium for a period of about 17 h. For tests of the HRG reactivity, the running buffer BSS HSA (SIGMA, Cat. No .: 05420) was added in a concentration of 1 mg / ml in order to avoid that peptides bind to the walls of the buffer interconnects. This was presented before the addition of HRG.

1.6.2 Chemosensitivity measurement

The cells were grown as described under 1.6.1 from culture bottles and on the polycarbonate membrane. Thereupon they remained in the incubator at 37 ° C. and 5% CO ₂ overnight. On the day of the examination, the medium was exchanged for a corresponding medium without FCS. The cells were left in this medium for 5 h before they were placed in the examination chambers.

The running buffer BSS with 100 units / ml was used for chemosensitivity measurements Penicillin G, 100 µg / ml streptomycin sulfate (LIFE TECHNOLOGIES, Cat. No .: 15140-122), 0.01 mg / ml kanamycin (SIGMA, Cat. No .: K 1377) and 2.5 µg / ml Amphotericin B (Fungizone, LIFE TECHNOLOGIES, Cat.No .: 15280-026) provided to prevent infections during the 15 h examination period check.

2 results 2.1 Heregulin stimulation

The functional detection of receptor overexpression is said to be with the help of the ligand HRG to be examined. HRG is a direct ligand for the HER3 and receptors HER4. At the signal generation induced by ligand binding and HER2 / neu is significantly involved in signal transduction (see section 1.4.4). The The answer is intended to shed light on the relative involvement of HER2 / new and its Give quantity and functionality.

Building on the findings of Chan et al. In 1995, cell lines were selected for examinations that differed with regard to the HER2 / neu, HER3 and HER4 receptors (Table 7). Table 7 Cell lines examined, receptor expression. Chan et al., 1995

2.1.1 Optimization of the measurement parameters

In order to develop a representative test procedure, the influence various factors that determine the measurement. These should then standardized for a uniform test procedure. The attempts were carried out on the cell lines listed in Table 6.

2.1.1.1 Cell density

Cells can differ in the metabolic activity measured in the cytosensor. Factors such as growth rate, cell size and time between plating and measurement play a role here. In order to make measurements with different cell lines comparable, a suitable cell density was first determined. The lower limit is defined by a minimum activity, which is necessary for the cytometric measurement. The measured activity should not be below 30 µV / s. At lower activities, the measurement error can reach critical values. In general, a cell density of 500,000 should not be exceeded, since otherwise the liquid flow, which transports protons to the reference electrode, is impeded by the large amount of cells. This shifts the typical acidification curve and the result is no longer reproducible (data not shown). To determine this suitable cell density, cells were plated at densities of 50,000, 100,000 and 300,000 cells / chamber and the metabolic activity was measured. There were large differences in activity related to cell density ( Fig. 8).

SK-BR-3 cells achieved a sufficient level with a cell density of 50,000 Activity of about 70 µV / s and were therefore used for further experiments in this density plated, while the other cells examined in a density between 200,000 (MDA-MB-453) and 300,000 (MCF-7, SK-OV-3) were plated. This Cells had a lower basic metabolism compared to SK-BR-3. If the cells were plated more than one night before the measurement, the Cell density chosen to be correspondingly lower.

2.1.1.2 Determination of the optimal heregulin dose

The second variable in the test approach is the HRG concentration. It became the ideal one Concentration determined from which a maximum reaction occurs. To this end the cells were exposed to various concentrations of HRG for 30 min. A concentration range from 1 ng / ml to 250 ng / ml was examined. For different cell lines resulted in different reaction profiles. For the HRG reactive cell lines, a minimum dose of 5 ng / ml was necessary to achieve a trigger verifiable reaction. From a concentration of 25 ng / ml approached the reaction quickly reached its maximum. In the area between these Concentrations were close to each other.

The saturation concentration of 100 ng / ml was defined as the standard stimulation concentration for further experiments. This concentration should ensure that the complexity of the functional effects can be demonstrated. Fig. 9 shows a measurement for dose-dependent HRG stimulation of the SK-BR-3 cell line. The arrow marks the addition of HRG.

Fig. 10 summarizes all evaluable measurements into a dose-response curve of this cell line. The maximum of the metabolic activity after stimulation is shown.

The MDA-MB-453 cell line shows a significantly lower level of stimulation. With the same stimulation duration and the same concentration range used, a maximum activity of approximately 130% is shown after stimulation. Since the control measurement also increases in the experiment shown ( Fig. 11), this value was subtracted from the value of the HRG stimulation curve at the corresponding point in order to conclude that the stimulation was effective. This results in a maximum stimulation of approx. 20% at 100 ng / ml HRG.

Although the dose-response curve of the MDA-MB-453 cell line shows a profile similar to that of the SK-BR-3 cell line, the response is significantly lower at all concentrations examined ( Fig. 12).

Fig. 13 shows a comparison of the dose-response curve of SK-BR-3 with that of MDA-MB-453. The stimulation with SK-BR-3 cells shows significantly higher values than is the case with MDA-MB-453 cells.

The MCF-7 cell line reacts only to a small extent to exposure to HRG ( Fig. 14).

The SK-OV-3 cell line is an ovarian cell line. Despite a clear HER2 / neu receptor amplification, this cell line shows no reaction to HRG ( Fig. 15).

2.1.1.3 Pacing Interval

For investigations on the dose-response curve based on investigations by Chan et al. a stimulation interval of 30 minutes was used. The course of the curve showed that the reaction reached the apex of some of the cell lines after a short time ( Fig. 9, 11). The influence of the exposure time on the reaction had to be investigated.

To determine the minimum exposure time that leads to a maximum response, were the HRG reactive cells with a constant HRG concentration of 100 ng / ml stimulated for different time intervals. A time window of 0 was used examined up to 28 minutes with an interval of 4 minutes.

The cells used showed clear differences in the kinetic receptor response. A complete reaction was observed with SK-BR-3 after approximately 12 minutes of exposure ( Fig. 16), while MDA-MB-453 required a minimum exposure of approximately 20 minutes ( Fig. 17). From this interval onwards, increasing the exposure does not increase the response rate. A stimulation time of 30 min should ensure in further experiments that a complete answer can proceed and was defined as the standard for further experiments. Fig. 16 and 17 show individual measurements of HRG stimulation depending on the exposure time. The arrow marks the beginning of the exposure.

The stimulation of the cell line MDA-MB-453 is very good for the experiment shown low. Therefore, no exact statement can be made. The maximum stimulation however occurs with exposure times over 20 min.

2.1.2 Herceptin inhibition of stimulation

As in numerous studies on signal transmission to EGF receptors Family described are in response to Heregulin alongside HER2 / new others Receptors involved (Beerli et al., 1995). For clear indications regarding the Share of HER2 / neu received in the HRG mediated response Herceptin inhibition experiments performed. For this purpose the HER2 / neu receptor specific with the monoclonal anti-HER2 / neu antibody Herceptin blocked and then stimulated the cells with 100 ng / ml HRG for 30 min and compared with the same stimulation without blocking HER2 / neu. Leaves a cell is stimulated by HRG despite Herceptin blockade of HER2 / neu, so this answer should represent the proportion that is not involved HER2 / new expires. First, different concentrations of the antibody were found used to determine the maximum concentration at which stimulation is inhibited.

The greatest observed metabolic activation by HRG was observed in the SK-BR-3 cell line. With a stimulation of over 40% at a concentration of 100 ng / ml HRG, these cells are suitable for the detection of the inhibition of this reaction by Herceptin. The response is partially inhibited even at an antibody concentration of 2 µg / ml. At a concentration of 4 µg / ml, the maximum inhibition is around 50% ( Fig. 18). This significant inhibition at 4 µg / ml Herceptin could be verified in repeated experiments and defined this concentration as the standard concentration for further experiments with Herceptin.

Fig. 19 summarizes several measurements for Herceptin inhibition for the cell line SK-BR-3. The percentage inhibition of the maximum stimulation without Herceptin is shown.

Fig. 20 shows that a certain minimum activation is necessary for a statement regarding the differentiation of the signal. A background noise of the series of measurements prevents statements that are below a range of approx. 10%. Nevertheless, it can be seen that in the experiment shown, no inhibition of HRG activation occurred at any of the Herceptin concentrations tested. Instead, a slight activation by blocking HER2 / neu was observed. The stimulation of 20% that was achieved in experiments on the dose-response curve could not be reconstructed at the time of the series of measurements for Herceptin inhibition. A dose-response curve was not created due to the mentioned inconsistency of the results.

2.1.3 Inhibition of the heregulin response by the tyrosine kinase inhibitor genistein

To investigate whether the route of the signal line initiated by HRG Tyrosine kinases were involved in investigating whether genistein stimulated one Can inhibit HRG reactive cell line.

For this purpose, SK-BR-3 cells were kept at 100 ng / ml for an interval of 30 minutes HRG and stimulated in parallel with 25 µg / ml genistein.

The stimulation in the presence of genistein was about 60% lower ( Fig. 21). This proves the involvement of tyrosine kinases in the signaling cascade triggered by HRG.

2.2 Chemosensitivity

To get results from the stimulation experiments with HRG with a response to be able to contact Herceptin Chemosensitivity measurements performed. This was to show whether Have verifications verified that indicate that certain cytostatics in Combination with Herceptin show additive or synergistic effects.

2.2.1 Selection of cytostatics

• - Cisplatin
• - Carboplatin
• - Vinorelbin
• - Gemcitabin
• - Paclitaxel

Cytostatics have been studied and are known to be potent in combination with Herceptin (Pegram et al., 1999a).

• - cisplatin
• - carboplatin
• - Vinorelbine
• - Gemcitabine
• - Paclitaxel

Additive or synergistic interactions have been particularly common to taxanes Paclitaxel and Docetaxel are described. For this purpose Chemosensitivity studies carried out. It turned out, however, that this approach Cell lines provide no information.

Taxanes are not water-soluble and act in a solvent dissolved in ethanol or DMSO. In chemosensitivity measurements, these solvents must be used as a negative control. It has been shown that cell lines are very sensitive to such solvents. The solvent reduced the metabolic activity in the experiments approximately as much as the cytostatic. Therefore no effect of the cytostatic could be derived. Figures 22 and 23 show chemosensitivity measurements of SK-BR-3 and MDA-MB-435 regarding paclitaxel.

Due to these strong effects, taxane studies were not carried out. In the illustration, an average was formed for a measurement period of 50 minutes. The corresponding value of the control measurement without active substance was subtracted from this value. In order to illustrate the ratio of this value to the control value, this value was added to 100. Values above 100% represent activation and values below 100% inhibition. In the chosen form of representation, the control measurement corresponds to 100% in every point. Each of the four cell lines was tested five times with the same substance. The cells were brought into contact with the cytostatic for 15 hours.

This representation applies if the control value is not decisively of 100 differs. This was the case with all measurements shown.

2.2.2 Chemosensitivity SK-BR-3

The SK-BR-3 cell line showed a mild response to cisplatin. The slight effect of 5% at the end measuring point of the curve could be improved by adding 10.5% to a total of 15.5% by adding Herceptin ( Fig. 24).

Carboplatin had no effect on the SK-BR-3 cell line. This substance led to a slight stimulation. No effect was found for the combination with Herceptin ( Fig. 25).

Vinorelbine showed the greatest effect among the substances tested in this cell line in combination with Herceptin. While no effect was observed for the mono substance, the combination with Herceptin led to an effect of approx. 15% in the last measurement point ( Fig. 26).

Gemcitabine had minimal effect on SK-BR-3 cells. This effect could be increased somewhat in combination with Herceptin ( Fig. 27).

Fig. 28 compares the results of the tested substances. In this diagram only the end points corresponding to the last 50 minutes of the measurement are taken into account. It is striking that vinorelbine only worked in combination with Herceptin, while cisplatin already had a slight effect as a mono substance.

For all substances tested, the combination with Herceptin led to one negative influence on cell metabolism. However, this effect was sufficient Carboplatin does not lack the slightly activating effect of the mono substance repealed. So there was no effect for this therapeutic be detected.

2.2.3 Chemical sensitivity MDA-MB-453

Cisplatin had no effect on the breast cancer cell line MDA-MB-453. The combination with Herceptin, on the other hand, showed a slight inhibition of metabolic activity ( Fig. 29).

The cytostatic drug carboplatin, as a single substance, had an effect similar to that of cisplatin + Herceptin. Herceptin was able to increase the cytostatic effect mediated by carboplatin by about 15% at the end point ( Fig. 30).

The result of the chemosensitivity test of vinorelbine indicated not only a good effect of the mono substance, but also a significant increase in the inhibition by adding Herceptin. The effect resulted in an effect of 20% and 35% for the combination ( Fig. 31). This result was very clear.

The effect that was achieved with the combination with Herceptin was most pronounced with vinorelbine. While no effect was found for the mono substance, the combination of vinorelbine with Herceptin achieved an inhibition of metabolic activity by more than 30% ( Fig. 32).

The studies of the cytostatic activity profile of the cell line MDA-MB-453 showed a very clear effect of Herceptin. In the case of gemcitabine, the addition of Herceptin increased the effect by over 30%, while in vinorelbine the effect of the mono substance could be increased by a further 15% from over 20% to over 35%. With all substances tested, an additional effect was shown by the combination of the substances with Herceptin ( Fig. 33).

2.2.4 MCF-7 chemosensitivity

Chemosensitivity studies of the MCF-7 cell line showed a cytostatic effect of approx. 10% for the substance cisplatin as a single substance, as well as in combination with Herceptin at the end point of the measurement. Herceptin did not influence the effect of this substance ( Fig. 34).

Carboplatin showed little cytostatic effect. This was increased by combining the cytostatic with Herceptin by approx. 5% to 10% in the end point ( Fig. 35). The additional effect is minor.

A slight effect was also observed with vinorelbine. A small effect of Herceptin increased the effect of this cytostatic from about 10% at the end point by a further 5% to 15%, which also corresponded to a small effect ( Fig. 36). Of the substances tested, vinorelbine had the greatest effect.

Gemcitabine had no effect on the MCF-7 cell line. The end point was 100% and thus corresponded to the value that the control measurement showed without the cytostatic agent. Herceptin had no effect ( Fig. 37).

The activity profile of the MCF-7 cell line states that the greatest observed effect comes from the cytostatics examined ( Fig. 38). Although a slight increase in the effect of Herceptin can be achieved with carboplatin and vinorelbine, this is clearly below the value that Herceptin triggers with HER2 / newly overexpressing cell lines SK-BR-3 and MDA-MB-453.

2.2.5 Chemical sensitivity SK-OV-3

Studies of the SK-OV-3 cell line were unable to demonstrate any effect of the cytostatic cisplatin. Combination with Herceptin also showed no inhibitory effect ( Fig. 39).

The substance carboplatin showed a similar result. While the mono substance had only a minimal negative effect on the metabolic activity of the cells after 15 hours, there was no effect for the combination with Herceptin ( Fig. 40).

The substance vinorelbine also had no cytostatic effect on the SK-OV-3 cell line. Here, too, Herceptin was unable to produce an additional effect, but even showed a slightly activating effect ( Fig. 41). Vinorelbine thus shows an antagonistic effect.

The substance gemcitabine could not negatively influence the metabolic activity of this cell line. Both the mono substance and the combination with Herceptin led to a slight stimulation ( Fig. 42).

In the SK-OV-3 cell line, none of the substances examined showed a cytostatic effect. Combining these substances with Herceptin also showed no effect ( Fig. 43).

2.3 Summary of results

The cell lines examined showed great differences in their response profile to HRG. Fig. 44 illustrates the different extent of the response to an exposure of 100 ng / ml HRG for an interval of 30 minutes. The largest response occurred with an average activation of 38% in the SK-BR-3 cell line. With an average activation of 19%, MDA-MB-453 cells show a lower, but still clearly detectable response. A minimal response of approximately 5% was found in the MCF-7 cell line, while no response could be measured in the SK-OV-3 cell line.

Chemosensitivity studies showed large differences in the cell lines in terms of their response to the substances tested. The HER2 / neu highly expressive cell lines SK-BR-3 and MDA-MB-453 showed a significantly increased effect when some of the substances tested were combined with Herceptin. The MCF-7 cell line, however, showed no increased effect of the combination of cytostatic and Herceptin in two out of four substances tested. In the other two cases, Herceptin showed minimal additive effects. For the SK-OV-3 cell line, effects that had a negative effect on metabolic activity could not be demonstrated for either of the cytostatics tested or for a combination with Herceptin (Table 8). Table 8 Herceptin enhances the effect of cytostatics after 15 hours of exposure

3 discussion 3.1 Background

The term "Theranostics" describes products that are therapeutic and possess diagnostic potential. A targeted diagnosis allows one Identify specific starting point for therapeutic agents and can thereby help a patient choose an optimal therapy. Because of big individual differences with regard to a therapeutic response there is great potential for cancer patients in oncology Development of "Theranostics" from. More and more therapeutic agents are doing it Pre-therapeutic examination of an expected response is necessary because these substances are increasingly causing problems for the population due to rising inflation rates Cause reimbursement. One deals in the field of cancer research deal with the identification of tumor markers, development and Automation of diagnostic techniques and clinical data collection, to characterize the prognosis and therapeutic outlook (Bromley, 2000).

The best known example of the use of "Theranostika" is HER2 / new, with which therapeutic antibody Herceptin. Evidence of an amplification of this Growth factor receptor grants a prognosis of an aggressive one Disease. This allows a statement to be made regarding an expected one In response to certain therapeutic agents. This is special crucial for the use of the anti-HER2 / neu antibody Herceptin (Cooke et al., 2001). The influence of diagnostics on therapy can be seen in this Example particularly clear, since in this case a positive proof of the Amplification of the receptor in the USA by the "Food and Drug Administration" (FDA) is a necessary criterion for admission to therapy with Herceptin. The aim of the work was to create an application-oriented and functional develop a diagnostic test that can identify the group of patients who from therapy with the monoclonal anti-HER2 / neu antibody Herceptin benefits. This test is said to have greater prognostic relevance than that established HER2 / new- approved by the "Food and Drug Administration" Test methods.

Herceptin was the first therapeutic monoclonal antibody to be used in therapy of metastatic breast cancer has been approved representative example of the specific approach of immunotherapy in the frame tumor therapy (Shepard et al., 1991). A frequent HER2 / neu amplification as The prerequisite for this form of therapy could be the breast (King et al., 1985), the colon (Meltzer et al., 1987), the lungs (Cline et al., 1987), the Magens (Jähne et al., 1994), the ovary (Meden et al., 1997), as well as the Pancreas (Buchler et al., 2001). This makes it suitable Therapy approach basically for the treatment of different tumors. The greatest scientific background on the monoclonal antibody However, Herceptin is in the field of gynecological tumors and here especially in the field of breast cancer. Clinical trials were both for Herceptin monotherapy, as well as for combination therapy with Cytostatics established in this area (Baselga et al. 1996). For this reason the investigations of the present work were carried out on cell lines that can be derived from breast and ovarian carcinomas.

A HER2 / new ampification is the prerequisite for an effect of Herceptin. Due to increasing development costs for future therapeutics and the associated higher and higher therapy costs can be assumed that in The future of such predictive test procedures will increasingly be run by patients are (Bromley et al., 2000). Treatment with Herceptin costs per year and Patient $ 36,000 (Taylor, 1999).

These surveys have already been discussed by experts at consensus meetings. Efforts to research a reliable prognostic test method were placed at the center of the discussion (Nelson et al., 2000).

The focus of the investigations of the present work is the HER2 / new Receptor, for the prognostic relevance in the diagnosis of breast Carcinoma (Cooke et al., 2001). In this work, the functional Analysis for this receptor and associated members of the EGFR family (Pinkas-Kramarski et al., 1996). The interaction of specific ligands, Antagonistic agents and antibodies acted as the basis for the elaboration a diagnostic test that serves as a template for future clinical studies was developed and optimized (Le et al., 2000). With this objective in mind "Theranostic" are developed, both prognostic, as well has a predictive character and differentiation and improvement so far established HER2 / neu test methods.

3.2 Establishment of a functional HER2 / neu receptor test

The detection of a receptor or gene amplification is the basis of the conventional and established HER2 / neu test methods. For this reason we first approached this approach with a functional focus experimentally. These investigations served primarily as a basis, a validation of the established procedures that take place on fixed tissue, now on vital To transmit cell systems.

HRG effects an agonistic stimulation on HER2 / neu, as previously Corresponding "ligand assays" has been described (Graus-Porta et al., 1997). in the We were interested in the context of this work, whether this effect can be Respond and about a change in the proton delivery of the tumor cell can be demonstrated. Therefore a dose escalation with HRG was taken as a basis and examined in detail.

It could be shown that the substance corresponds to the receptor density provides a consistent answer that accompanies dose escalation. Based on Investigations by Chan et al. 1995 stimulation experiments with the specific ligand HRG to make a statement about HER2 / neu- To hit receptor density on the cell membrane. The signal line passing through Binding of this ligand is involved, includes HER2 / neu. Lewis et al. were able to show in 1996 that HER2 / neu induced a response in the HRG plays a crucial role. These studies have shown that HER2 / new is essential in triggering a proliferative response and only this takes place when HER2 / new is available. For a reactivity towards HRG are on the one hand, the receptors HER3 and HER4 as direct interaction partners for HRG necessary, as well as HER2 / new for an effective signal line. Research by another group showed that HER2 / new for the others Members of the EGFR family used the preferred receptor in the ligand-induced Represents heterodimerization (Graus-Porta et al., 1997).

The results of Chan et al. 1995 to confirm. So all cell lines reacted with an increase in metabolic Activity on an HRG exposure that on the one hand contains at least one of the HRG Receptors (HER3, HER4) and on the other hand an amplification of HER2 / new Had receptor (SK-BR-3, MDA-MB-453). The cell line that named the HRG receptors were missing (SK-OV-3), as well as those in which no HER2 / new Amplification could be detected (MCF-7), could not or hardly stimulate (Chan et al., 1995; De Fazio et al., 2000). Quantitative differences in a stimulation could be made with the relative amount of expressed HER2 / neu receptors can be associated. Proceeding from this the influence of the test parameters on HRG stimulation is examined and the Experimental approach can be optimized. As a result, a clearer Increase in activation can be achieved when in 1995 by Chan et al. reached has been.

The results show that the test approach represents one possibility, one To make a statement regarding the EGF receptor distribution, the additional clues regarding the amount of HER2 / neu receptors on the cell membrane examined cells considered.

An extension of this test approach should show whether tyrosine kinases are involved in the signal line induced by HRG. An inhibition with the help of the tyrosine kinase inhibitor genistein effectively minimized the triggered reaction ( Fig. 21). This demonstrates the dominant function of tyrosine kinases on the signaling pathway induced by HRG. The development of specific tyrosine kinase inhibitors could lead to a further development of this test approach allowing more specific conclusions.

3.3 Evidence of specific interactions between Herceptin and HER2 / new

Another question that arises when examining the HER2 / neu receptor was investigated whether the described approach of the functional Receptor tests also specific interactions of the monoclonal antibody Have Herceptin proven.

To prevent such interactions by Herceptin with the HER2 / neu receptor investigate the influence of Herceptin in interaction with HRG examined. Anti-HER2 / neu antibodies have been described to affect the stability of HER2 / neu-HER3- as well as HER2 / neu-HER4 heterodimers impair and thereby accelerating ligand dissociation (Klapper et al., 1997). This Competition of the monoclonal antibody Herceptin with the agonistic Ligand HRG to bind HER2 / neu is a mechanism for which Effect of Herceptin is blamed (Baselga et al., 2001b).

The expectation that the HRG-induced activation is inhibited by blocking HER2 / neu with the antibody was confirmed in the cell line SK-BR-3. Thus, the stimulation with HRG decreases when the cells are exposed to Herceptin depending on the concentration of the monoclonal antibody less than in control experiments that were performed without adding the antibody. Herceptin was unable to completely suppress the reaction ( Fig. 18). This could be an indication that the interaction of HRG with the receptor is not competitive on its own and that a proportion of the receptors are still involved in a signal line despite blocking. Another explanation would be that the HER2 / neu blockade involves other receptors such as the EGFR for heterodimerization.

Graus-Porta et al. published a model in 1997 that states that the HRG HER3 and HER4 receptors prefer to interact with HER2 / neu, in the absence from HER2 / new but also with EGFR. According to this model, one could Reaction that occurs despite blocking the HER2 / neu receptor, thus explaining that alternatively to the preferred dimerization of the HRG receptors with HER2 / new an interaction of these receptors with the EGFR can occur and thereby A signal can be generated in the form of a stimulation of the metabolism is detectable. Comparable studies were carried out in 1996 by Lewis et al. carried out. They examined the influence of monoclonal anti-HER2 / new Antibodies to tyrosine phosphorylation by HER2 / neu that are associated with exposure with HRG. It was shown that this phosphorylation by blocking with the help of the antibody by about 50%.

Attempts to Herceptin inhibition of HRG stimulation on the MDA-MB-453 cell line proved to be of little value. The stimulation achieved in section 2.1.1.2 could not be reproduced for these experiments. There was no evidence of inhibition. When interpreting the results for the MDA-MB-453 cell line, it must be taken into account that the reaction lies in a measuring range in which the measurement error is relatively large in relation to the reaction. Differences in the measurement curves obtained can only be approximated ( Fig. 20).

The other cell lines examined in this work showed little or no evidence detectable response to HRG. The study of Herceptin interaction These cell lines confirmed the finding that these cells were not compared to HRG are reactive. A change in this reaction profile by blocking the Herceptin's HER2 / neu receptor could not be determined (data not ) Shown.

Further insight into the relationships between receptor interactions and their Effects on the kinetics of a response to HRG could be seen in further experiments are obtained by other receptors of the EGF family through antibodies be blocked. It would be expected that the EGFR would be blocked in Combination with the blocking of the HER2 / neu receptor into one leads to complete suppression of the HRG-induced response.

3.4 Studies on the combination of Herceptin with cytostatics

The results for the examined cell lines were validated by Chemosensitivity measurements. Basis of this microphysiometric analysis is the diagnostic test procedure ChemoSelect from CELLCONTROL AG. This procedure allows tumor cells to come into contact with various cytostatics to be brought. A measurement of the cell metabolism provides information about one Effect of the substances tested. For this diagnostic test procedure a good in vitro / in vivo correlation has already been demonstrated (Metzger et al., 2001).

While Herceptin was initially administered as a monotherapy, it is used today Combined with cytostatics. To validate the above The question was to design test results as application-related as possible a response to Herceptin. Additive or synergistic effects Herceptin with these cytostatics should be investigated. To do this Cytostatics were investigated in which such interactions have been demonstrated (Baselga et al., 1998; Pegram et al., 1999a; Baselga et al., 2001, Nabholtz et al., 2001).

The method of chemosensitivity measurement can add to that Statement about a response to Herceptin an indication of the optimal Combination of cytostatic and Herceptin take place. On this point one offers functional test procedure a significant improvement over the established ones HER2 / neu tests, which in the clinic problems of the choice of therapy encountered.

Chemosensitivity studies showed that the SK-BR-3 and MDA-MB-453 an additional effect of Herceptin in combination with revealed some of the cytostatics examined. A therapeutic effect could thus with these cell lines. This therapeutic "benefit" is in these Cases correlated positively with evidence of reactivity to HRG and confirms the hypothesis that if stimulation by HRG occurs Response to Herceptin is expected. These results are therefore in Consistent with the assumption of HER2 / neu overexpression as necessary, however insufficient criteria for a response to Herceptin.

For the cell lines that could not be stimulated by HRG (MCF-7, SK-OV-3), could not have any therapeutic benefit with the combinations of cytostatics Herceptin can be detected. Nevertheless, one could be found for some cytostatics Determine effect, which is not or only minimally enhanced by Herceptin could be. A response to Herceptin was therefore not possible with these cells to be watched. In these cases too, the HRG reactivity is positive with a Response to Herceptin correlated.

The MCF-7 cell line has an EGF receptor assembly that already no response can be expected with the established HER2 / neu test methods. A Overexpression of the HER2 / neu receptor cannot be found and therefore cells of this type would also use the established HER2 / neu test methods "Non-responders" classified (Chan et al., 1994). The cell line SK-OV-3 shows however, significantly amplified values with regard to this receptor. For these cells however, no therapeutic benefit of Herceptin has been demonstrated, though these cells are eligible for therapy according to the FDA criteria. Consequently these cells seem to represent a case in which using established methods incorrectly responded to because of a positive HER2 / neu test Herceptin would shut down. This case is representative of 75-90% of the HER2 / newly positive tested patients who are not on treatment with Address Herceptin (Baselga et al., 1996).

The results obtained indicate that the reactivity investigated has a correlation with HRG with the response to Herceptin. This conclusion has to be drawn, as shown in the examined tumor cell models that Herceptin was only effective in cells via HER2 / neu amplification and additionally over a certain amount HER3 and HER4 on the cell membrane. The results of the Chemosensitivity measurements prove a prognostic relevance, which A significantly improved comparison to the established HER2 / neu test methods Enable predictive value.

3.5 Outlook

Numerous efforts are being made to determine criteria according to which tumor patients can be guaranteed optimal therapy. The Much of the research is concerned with the molecular biological detection of Tumor markers at the genetic level. So one hopes through one standardized "screening" of several thousand genes a genetic profile to define that characteristic of chemosensitivity to certain Substances is (Staunton et al., 2001). For such approaches, however, there is insufficient clinical data at this time. To a final one To be able to make a statement regarding the predictive value of such procedures, this data must be collected.

Functional "assays" offer a completely different approach. In contrast to molecular biological applications these techniques deal with effects, which are reproduced by functional cells in a physiological environment become. So proliferation models offer an approach that has functional effects based on the proliferation rate of tumor cells. Animal models can also be used make a certain statement about the response of transplanted cells. However, both methods are complex and have a long measurement duration (days to Weeks) until a statement can be made. This time is conditional through the necessary phases of cell culture.

• 1. Bezüglich der hier untersuchten Zelllinien kann prognostische Relevanz erreicht werden, die deutlich bessere Ergebnisse erzielt, als dies mit den etablierten HER2/neu-Testverfahren möglich ist.
• 2. Da der Test im Optimalfall innerhalb von 24 Stunden nach Entnahme der Biopsie abgeschlossen ist, kann die Therapie bereits sehr bald begonnen werden.
• 3. Eine Veränderung von Tumorzellen vor der Testung (z. B. auftretende Resistenzen), wie sich bei Testverfahren mit notwendiger Zellkulturphase vorkommen kann, stellt für dieses Verfahren kein Problem dar.
• 4. Da der funktionelle Rezeptortest Effekte an lebenden Zellen misst, die der in- vivo Situation nahe kommen, werden besonders Faktoren berücksichtigt, die an der in-vivo-Reaktion beteiligt sind.

The functional test approach examined in this work offers ideal conditions for developing a competitive product that is tailored to the needs of the market. The test offers the following advantages over alternative methods:

• 1. With regard to the cell lines examined here, prognostic relevance can be achieved, which achieves significantly better results than is possible with the established HER2 / neu test methods.
• 2. Since the test is optimally completed within 24 hours after the biopsy is taken, the therapy can be started very soon.
• 3. A change in tumor cells prior to testing (eg resistance that occurs), as can occur in test methods with a necessary cell culture phase, is not a problem for this method.
• 4. Since the functional receptor test measures effects on living cells that come close to the in vivo situation, special attention is paid to factors that are involved in the in vivo reaction.

To ultimately validate the test results obtained based on clinical data can, analogous examination must be carried out on cells that come out immediately Tumor tissue originate. The disadvantage of a possibly additional operational Intervention stands for the patient a high security when choosing an optimal one Therapy versus. To the knowledge gained on cell lines on biopsies The parameters of the test that have already been worked out can be coordinated with them Needs to be adjusted.

A crucial step in the examination of tissue cells in the Compared to experiments on cell lines, there is tissue processing. This step is dominated by digestion with enzymes and can take Possibly non-specific effects on the extracellular receptor domains cause. However, there is already experience with the Dissociation methods deviating from the standard processing protocol. A A suitable processing method could be, for example, a mild one Enzyme combinations or gentle mechanical procedures can be performed. However, problems with the provision of tumor cells are not expected. Experience of the company CELLCONTROL AG in the area of logistics and Supervision of clinical studies as part of the development of the ChemoSelect test, will be useful to ensure a smooth procurement process To ensure tumor cells.

An important class of substances for which additional effects in combination with Herceptin have been described in clinical studies are taxanes (Slamon et al., 2001). Initial chemosensitivity studies of these substances showed that these measurements do not allow any conclusions to be drawn about an effect. This can be explained by the fact that these substances are not water-soluble and therefore solvents have to be added in the corresponding negative control tests. These solvents showed strong toxicity to the cell lines examined. As a result of these non-specific solvent effects, the metabolic activity of the cells was already reduced so much by the solvent that it was not possible to derive an effect of the investigated cytostatic in these cases ( Fig. 22, 23). Therefore, studies with these cytostatics had to be avoided.

For investigations of chemosensitivity, cytostatics were used according to their "Peak plasma" concentration used. This concentration is to the situation in Get close to the body during therapy. One approach to the problem of toxicity To encounter solvents would be in a correspondingly low concentration to use. A series of tests could provide information as to whether a the unspecific concentration of the cytostatics chosen accordingly low Effects are to be minimized.

An extension of the test procedure is with other ligands for the EGFR family imaginable, which also HER2 / new with the signal line include. For example, the reaction profile of EGF or others HRG isoforms are examined. In this way, the prognostic value of the statement of a stimulation by another Substance or even a "cocktail" of various substances further improved become.

An application of this test approach could be used to study tumors other organs. There are more prerequisites for such an approach clinical data on Herceptin response.

Further cellular "targets" of the tumor will be of interest in the future: couple via comparable signal transmission paths, as is the case with EGF receptors to do. Substances are already in early clinical validation today cause a specific blockage of the intracellular signal-effector cascade. These active ingredients are the beginning of a specific and future-oriented Therapy, the effectiveness of which is based on modern, functional diagnostic Test procedure is confirmed to ensure an individual response. The The test on which this work is based has created the first prerequisites for this, review and confirm use in clinical trials.

4 Summary

The "Epidermal Growth Factor" receptors (EGFR) represent potential "targets" for is tumor therapy. HER2 / neu is a member of this receptor family and will used as a prognostic marker in breast cancer. Against this receptor develops a humanized, monoclonal antibody (trastuzumab, Herceptin), the first approved for clinical applications in tumor therapy Represents antibodies and triggers a specific inhibition of the tumor cell. This is used in breast cancer patients who have a high HER2 / neu density exhibit (at about 25%). Although Herceptin only after diagnostic confirmation administered to HER2 / new positive patients still speak less than 30% of patients responded to therapy with the antibody. Therefore Herceptin in combination with certain cytostatics as a combination therapy administered to improve response. Still benefit many HER2 / new-positive patients fail from the actual immunotherapy. Therefore, there is a need for a test procedure. A functional Herceptin Test could solve this problem and identify "therapy responders". In addition, such a test could also be the synergistic substance partner of one Identify combination therapy.

In the first part of this work, such a functional test procedure was established. This test can activate the HER2 / neu receptor through the ligand Heregulin and the selective and antagonistic effects of Herceptin on it Prove activation. For this, different cell lines of the breast and the Ovars used, which had different characteristics. It was shown that activation increased with receptor density and differential in vitro "Response rates" were achieved for the different cell types. In the Ranking SK-BR-3 >> MDA-MB-453 >> MCF7 took effect with the ligand analogous to the receptor density. Expression of all was of all cell types HER3 receptors in common. It was also proven that the Presence of a "mediator molecule" which is another specific Receptor subtype is the prerequisite for a functional response. SK In the absence of HER3, OV-3 shows a similarly high receptor density as SK-BR-third A stimulatory response could not be proven with Heregulin, since the lack of HER3 as a cofactor has no functional response in this Permitted cell type In the combination Heregulin and Herceptin the inhibitory effects of Herceptin can be demonstrated. Herceptin also indicates functionally decoupled SK-OV-3 cells have no effect.

The synergism in the sense of selective inhibition of the tumor cells was eliminated a combination therapy of Herceptin and five different cytostatics predictive value examined. It could be shown that only the activatable cell lines (see above) a selective response to the additive administration of Herceptin and cytostatics were admitted, while the SK-OV-3 cell lines had no effects exhibited. Furthermore it could be proven that individual differences in the cell lines led to different degrees of co-inhibition. It was able to respond to the effectiveness of Herceptin Cytostatics, shown in the sense of demonstrable sensitivity or resistance become.

These findings confirm that this test system in the future, for which Determination of the patient population could be positioned to "Herceptin Responder "of" non-responder "before an upcoming therapy functional and to be able to differentiate quickly. At the same time, this test system leaves an accurate Determination of the synergistic and antagonistic cytostatics on the Tumor biopsies of the patient.

This work forms the basis for a "theranostic" procedure with which in the future treatment with Herceptin can for the first time be focused specifically on patients who respond to the drug. This would lead to a significant reduction in side effects and costs of therapy. Continued work with patient biopsies and clinical trials must now determine the quality of this system. 5 Bibliography Alroy, I., Yarden, Y. The erbB signaling network in embryogenesis and oncogenesis: Signal diversification through combinatorial ligand-receptor interactions. FEBS Lett 410: 83-86 (1997);
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Illustration illustration
ATP: adenosine 5'-triphosphate
BSS: Balanced salt solution
CaCl ₂ : calcium chloride
CDR: Complementary determining region
CHO: Chinese hamster ovary
CM: Cytosensor microphysiometer
There: Dalton
DMSO: dimethyl sulfoxide
ECD: Extracellular domain
EDTA: ethylenediaminetetracetic acid disodium salt dihydrate
EGF: Epidermal growth factor
EGFR: Epidermal growth factor receptor
EGTA: ethylene glycol bis (2-aminoethyl) tetraacetic acid
ER: estrogen receptor
EtOH: ethanol
FDA: Food and Drug Administration
FISH: Fluorescence in situ hybridization
HER2 / new: Human epidermal growth factor receptor 2
HER3: Human epidermal growth factor receptor 3
HER4: Human epidermal growth factor receptor 4
HRG: Heregulin
HSA: Human Serum Albumin
Ig: immunoglobulin
IgG1: immunoglobulin G1
IHC: Immunohistochemistry
KCl: potassium chloride
LAPS: Light addressable potentiometric sensor
M: Molar
MgCl ₂ : magnesium chloride
ml: milliliters
mM: millimolar
mRNA: messenger RNA
Na ₂ HPO ₄ : disodium hydrogen phosphate
NaCl: sodium chloride
NaH ₂ PO ₄ : sodium dihydrogen phosphate
NaHCO ₃ : sodium pyruvate
NaOH: sodium hydroxide
NDF: New differentiation factor
ng: nanogram
NRG: Neuregulin
PBS: Phosphate buffered saline
PBS: Phosphate buffered saline solution
PCR: polymerase chain reaction
RTK I: Class I receptor tyrosine kinase
RTK: receptor tyrosine kinase
s: second
Tab: table
Tris: tris (hydoxymethyl) aminomethane
µg: microgram
uV:

Claims (38)

1. Comprehensive methods for predicting or predicting the effectiveness of tumor treatment a) contacting a sample that has living cells with abnormal activity of a member of the EGF receptor family a) an interaction partner, the interaction partner modulating the signal of the receptor mediated by the natural ligand; and b) one of several therapeutic agents under conditions that allow the living cells to interact with the interaction partner and the one or more therapeutic agents; and b) Measurement of the signal mediated by the member of the EGF receptor family, the modulation of this signal predicting or predicting the effectiveness of the tumor treatment. 2. The method of claim 1, wherein the modulation of the receptor-mediated Signal in a living cell with abnormal activity of a member the EGF receptor family with the modulation of the corresponding and under the same process conditions brought about signal in one living cell with normal activity of the member of the EGF receptor family is compared and the effectiveness of tumor treatment by the Difference in the modulation is predicted or predicted. 3. The method according to claim 1 or 2, wherein the therapeutic agent is selected from a group consisting of a substance with toxic effects, an ionic emitter, an antibody and a hyperthermic Therapeutic. 4. The method of claim 3, wherein the therapeutic agent is a combination of two or more therapeutic agents. 5. The method of claim 3, wherein the interaction partner and the Therapeutic are fused together. 6. A method for identifying a therapeutic agent effective for tumor treatment or a lead substance for such a therapeutic agent a) contacting a sample that has living cells with abnormal activity of a member of the EGF receptor family a) an interaction partner, the interaction partner modulating the signal of the receptor mediated by the natural ligand; and b) one or more test substances under conditions that allow the living cell to interact with the interaction partner and the test substance; a) measurement of the signal mediated by the member of the EGF receptor family; and b) Comparison of the measurement result from step (b) with a measurement result which was obtained without the addition of the test substance, the modulation of this signal with the test substance compared to the value without the test substance being an indication of the effectiveness of the test substance as a therapeutic agent or as a lead substance for a therapeutic agent. 7. The method according to any one of claims 1 to 6, wherein the interaction partner is a monoclonal antibody or a fragment or derivative thereof. 8. The method of claim 7, wherein the monoclonal antibody is an IgG. 9. The method of claim 7 or 8, wherein the monoclonal antibody is humanized antibody. 10. The method of claim 9, wherein the humanized antibody Herceptin (Trastuzumab) is. 11. A method for identifying a therapeutic agent effective for tumor treatment or a lead substance for such a therapeutic agent comprising a) contacting a sample that has living cells with abnormal activity of a member of the EGF receptor family a) one or more tumor therapeutics; and b) an interaction partner to be tested for efficacy in tumor treatment, the interaction partner modulating the signal of the receptor mediated by the natural ligand under conditions that allow the living cell to interact with the tumor therapeutic and the interaction partner; b) measurement of the signal mediated by the member of the EGF receptor family; and c) Comparison of the measurement result from step (b) with a measurement result that was obtained without the addition of the interaction partner, the modulation of this signal with the interaction partner compared to the value without the interaction partner being an indication of the effectiveness of the interaction partner as a combination therapeutic agent or as a lead substance for a combination therapeutic. 12. The method according to any one of claims 6 to 11, wherein the modulation of the Receptor-mediated signal in a living cell with an abnormal Activity of a member of the EGF receptor family with the modulation of the corresponding signal in a living cell with normal activity of the Members of the EGF receptor family are compared and the modulation of these Signals an indication of the effectiveness of the therapeutic agent or Combination therapeutic agent or a lead substance for such represents. 13. The method according to any one of claims 1 to 12, wherein the abnormal activity Is the result of overexpression of the receptor. 14. The method according to any one of claims 1 to 13, wherein the interaction partner intrinsic potential for modulation of the receptor-mediated signal has. 15. The method according to any one of claims 1 to 14, wherein the interaction partner is an agonist of the receptor-mediated signal. 16. The method according to any one of claims 1 to 14, wherein the interaction partner is an antagonist of the receptor-mediated signal. 17. The method according to any one of claims 1 to 16, wherein the member of the EGF Receptor family is the HER2 receptor. 18. The method according to any one of claims 1 to 17, wherein the sample is a cell or tissue sample. 19. The method of claim 18, wherein the cell or tissue sample is a Tumor sample is. 20. The method of claim 19, wherein the tumor sample is a sample of solid tumor or from metastatic cells made by a solid Tumors are descended. 21. The method of claim 20, wherein the solid tumor is selected from a group including breast cancer, ovarian cancer, Bronchial carcinoma, colon carcinoma, melanoma, bladder carcinoma, Gastric carcinomas, head / neck tumors, brain tumors, cervical tumors, Prostate cancer, testicular cancer, swanoma, leiomyosarcoma, Bone tumors, renal carcinomas, pancreatic tumors and Esophageal tumors. 22. The method of claim 19, wherein the tumor sample is a sample of Tumor is of hematopoietic origin. 23. The method of claim 22, wherein the tumor is of hematopoietic origin is selected from a group comprising malignant lymphomas, leukemic cell, non-Hodgkin lymphoma, Hodgkin lymphoma and Thyroid lymphomas. 24. The method of claim 22 or 23, wherein the tumor sample is a sample of tumor cells circulating in the blood. 25. The method according to any one of claims 1 to 24, wherein the modulation of the Receptor-mediated signal is measured as induction of cell death or Proliferation or inhibition of proliferation. 26. The method according to any one of claims 1 to 25, wherein the measurement in one functional assay. 27. The method of claim 26, wherein the functional assay is selected from a group comprising proliferation assays, cytotoxicity assays and metabolic assays. 28. The method according to any one of claims 6 to 27, wherein the therapeutic agent or combination therapeutic or the lead substance for such Therapeutic agent a small molecule, a peptide, aptamer, Is antibody or fragments or derivatives thereof. 29. The method according to any one of claims 6 to 28, the one Is high throughput. 30. The method of claim 29, which is computer-assisted. 31. A method for improving the pharmacological properties of the therapeutic or combination therapeutic identified by a method according to any one of claims 6 to 30 or the lead substance for such a therapeutic, wherein a) identifies the binding site of the therapeutic agent or the lead substance for such a therapeutic agent to the receptor, to a receptor-associated coreceptor or to a signal-related molecule that is related to the signal of the receptor; b) the binding site of the therapeutic agent or the lead substance for such a therapeutic agent to the receptor, to a receptor-associated coreceptor or to a signal-related molecule which is related to the signal of the receptor and modified by molecular modeling; and c) the therapeutic agent or the lead substance for such a therapeutic agent is modified in such a way that its binding specificity or binding affinity or binding avidity for the receptor, a receptor-associated coreceptor or a signal-mediating molecule related to the signal of the receptor is increased by molecular modeling. 32. The method of claim 31, wherein the binding sites in step (a) by Position-specific mutagenesis can be determined. 33. The method according to any one of claims 28 to 32, wherein the identified or improved the therapeutic or the lead substance for such Therapeutic agent further improved pharmacologically through peptidomimetics becomes. 34. The method according to any one of claims 6 to 33, wherein the therapeutic agent or the lead substance for such a therapeutic agent is further modified to a) a modified active center, a modified activity spectrum, a modified organ, tissue or cell specificity; b) improved activity; c) increased toxicity to tumor cells (an improved therapeutic index); d) reduced side effects; e) a delayed start of therapeutic effectiveness, the length of therapeutic effectiveness; f) changed pharmacokinetic parameters (absorption, distribution, metabolism or excretion); g) modified physicochemical parameters (solubility, hygroscopic properties, color, taste, smell, stability, condition); h) improved general specificity, organ / tissue specificity; and or i) optimized administration form and route by a) esterification of carboxyl groups or b) esterification of hydroxyl groups with carboxylic acids or c) esterification of hydroxyl groups to, for example, phosphates, pyrophosphates or sulfates or "hemisuccinates" or d) formation of pharmaceutically acceptable salts or e) formation of pharmaceutically acceptable complexes or f) synthesis of pharmacologically active polymers or g) introduction of hydrophilic groups or h) introduction / exchange of substituents in aromatics or side chains, change of the substituent pattern or i) modification by introduction of isosteric or bioisosteric groups or j) synthesis of homologous compounds or k) introduction of branched side chains or l) conversion of alkyl substituents to cyclic analogs or m) derivatization of hydroxyl groups to ketals or acetals or n) N-acetylation to amides, phenyl carbamates or o) Synthesis of Mannich bases, imines or p) Conversion of ketones or aldehydes to ship bases, oximes, acetals, ketals, enolesters, oxazolidines, thiozolidines or combinations thereof. 35. Process for the manufacture of a medicament or medicinal product, wherein one obtained by the method according to any one of claims 6 to 34 Therapeutic or the lead substance for such a therapeutic with a pharmaceutically acceptable carrier or diluent formulated. 36. The method according to any one of claims 6 to 35, wherein the therapeutic agent or combination therapeutic or the lead substance for such Therapeutic is a cytostatic or a cytotoxic substance. 37. Use of a method according to one of the preceding Claims identified therapeutic or combination therapeutic for the manufacture of a drug or medical device for treatment of tumors and or tumor diseases. 38. Medicaments containing one or more of the therapeutic agents which are used after one Method according to one of claims 6 to 36 identified or improved were.